Immunocompatible amniotic membrane products

ABSTRACT

Provided herein is a placental product comprising an immunocompatible amniotic membrane. Such placental products can be cryopreserved and contain viable therapeutic cells after thawing. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.

RELATED APPLICATIONS

This application claims priority to:

U.S. Provisional Applications Ser. No, 61/338,464 entitled “Selectively Immunodepleted Chorionic Membranes” , filed on Feb. 18, 2010 bearing Docket No. 22924US01,

U.S. Provisional Applications Ser. No, 61/338,489 entitled “Selectively Immunodepleted Amniotic Membranes”, filed on Feb. 18, 2010 bearing Docket No. 22925US01, and

U.S. Provisional Applications Ser. No, 61/369,562 entitled “Therapeutic Products Comprising Vitalized Placental Dispersions filed on Jul. 30, 2010 bearing Docket No 23498US01, the contents of which are hereby incorporated by reference in their entireties.

This application is being co-filed on Feb. 18, 2011 with, and incorporates by reference, applications entitled:

“Methods of Manufacture of Immunocompatible Chorionic Membrane Products”,

“Immunocompatible Chorionic Membrane Products”,

“Immunocompatible Amniotic Membrane Products”,

“Methods of Manufacture of Immunocompatible Amniotic Membrane Products”,

“Therapeutic Products Comprising Vitalized Placental Dispersions” , and

“Methods of Manufacture of Therapeutic Products Comprising Vitalized Placental Dispersions”

FIELD OF THE INVENTION

The present technology relates to products to facilitate wound healing such as placenta membrane-derived products and biologic skin. The present technology relates to products to protect injured or damaged tissue, or as a covering to prevent adhesions, to exclude bacteria, to inhibit bacterial activity, or to promote healing or growth of tissue. An example of such a placental membrane is an amniotic membrane. The field also relates to methods of manufacturing and methods of use of such membrane-derived products.

BACKGROUND OF THE INVENTION

Fresh or decellularized placental membranes have been used topically in surgical applications since at least 1910 when Johns Hopkins Hospital reported the use of placental membrane for dermal applications. Subsequently unseparated amnion and chorion were used as skin substitutes to treat burned or ulcerated surfaces. During the 1950′s and 60's Troensegaard-Hansen applied boiled amniotic membranes to chronic leg ulcers.

The human amniotic membrane (AM) is the innermost of the fetal membranes deriving from the amniotic sac and constituting the lining of the amniotic cavity. It is approximately 0.02 to 0.5 mm thick. The AM consists of five layers: a thin layer rests on the basement membrane and contacts the amniotic fluid, an underlying layer of connective tissue attaching the basement membrane that consists of three layers: a compact layer, a layer of fibroblast, and a spongy layer. The spongy layer is adjacent to the cellular layer of the chorion. The amnion is essentially devoid of vasculature.

Both fresh and frozen AMs have been used for wound healing therapy. When fresh AM is used, there is increased risk of disease transmission. According to published reports, fresh amniotic tissue exhibits cell viability of 100%, however within 28 days of storage above 0° C. diminished cell viability to 15 to 35%. Freezing over a time of 3 weeks reduced cell viability to 13 to 18%, regardless of the temperature or medium.

Lee and Tseng report the successful cryopreservation of AM in glycerol and Dulbeccos Modified Eagle medium (DMEM) at −80° C., although such cryopreservation dramatically decreases cell viability. The cryopreservation of AM in glycerol and DMEM is recommended by the FDA. According to published reports, glycerol storage of AM resulted in immediate cell death. Glycerol cryopreserved AM (−80° C.) and glycerol-preserved AM (−4° C.) are sufficient to provide a matrix for wound healing, but fail to provide sufficient cell viability to bestow biological effectiveness

Gajiwala and Gajiwala report the successful preservation of AM by freeze-drying (lyophilisation) and gamma-irradiation. According to this method, AM is pasteurized at 60° C., treated with 70% ethanol, and freeze-dried to remove most of the remaining moisture. Then the AM is sterilized by exposure of 25 kGy gamma-radiation in a Cobalt 60 Gamma chamber unit or at an ISO-certified radiation plant. The sterilized AM can be stored at room temperature for a short period (up to 6 months).

Gomes reports preservation of AM with lyophilisation followed by sterilization in ethylene oxide.

Rama et al reported the cryopreservation of AM in 10% dimethyl sulfoxide (DMSO) instead of glycerol and achieved a cell viability of about 40%.

Two placental tissue graft products containing living cells, Apligraf and Dermagraft, are currently commercially available. Both Apligraf and Dermagraft comprise ex vivo cultured cells. Neither Apligraf nor Dermagraft comprise stem cells. Furthermore, neither Apligraf nor Dermagraft comprise Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) and adiponectin, which are key factors in the natural wound healing process. In addition, neither Apligraf nor Dermagraft exhibit a protease-to-protease inhibitor ratio favorable for wound healing. As wound healing is a multi-factorial biological process, many factors are needed to properly treat a wound; products having non-native cellular populations are less capable of healing wounds relative to a product having an optimal population of cells representing the native array. It would represent an advance in the art to provide a chorion-derived biologic skin substitute comprising a population of cells representing the native array of factors, including, for example, growth factors and cytokines.

Apligraf is a living, bi-layered skin substitute manufactured using neonatal foreskin keratinocytes and fibroblasts with bovine Type I collagen. As used in this application, Apligraf refers to the product available for commercial sale in November 2009.

Dermagraft is cryopreserved human fibroblasts derived from newborn foreskin tissue seeded on extracellular matrix. According to its product literature, Dermagraft requires three washing steps before use which limits the practical implementation of Dermagraft as a wound dressing relative to products that require less than three washing steps. As used in this application, Dermagraft refers to the product available for commercial sale in November 2009.

Engineered wound dressings such as Apligraf and Dermagraft do not provide the best potential for wound healing because they comprise sub-optimal cellular compositions and therefore do not provide proper wound healing. For example, neither Apligraf nor Dermagraft comprises stem cells and, as a result, the ratio between different factors secreted by cells does not enable efficient wound healing. Additionally, some factors that are important for wound healing, including EGF, IGFBP1, and adiponectin, are absent from both Apligraf and Dermagraft. Additionally, some factors, including MMPs and TIMPs, are present in proportions that differ greatly from the proportions found in the natural wound healing process; this difference significantly alters, among other things, upstream inflammatory cytokine pathways which in turn allows for sub-optimal micro-environments at the wound site.

Paquet-Fifield et al. report that mesenchymal stem cells and fibroblasts are important for wound healing (J Clin Invest, 2009, 119: 2795). No product has yet been described that comprise mesenchymal stem cells and fibroblasts.

Both MMPs and TIMPs are among the factors that are important for wound healing. However, expression of these proteins must be highly regulated and coordinated. Excess of MMPs versus TIMPs is a marker of poor chronic wound healing (Liu et al, Diabetes Care, 2009, 32: 117; Mwaura et al, Eur J Vasc Endovasc Surg, 2006, 31: 306; Trengove et al, Wound Rep Reg, 1999, 7: 442; Vaalamo et al, Hum Pathol, 1999, 30: 795).

α2-macroglobulin is known as a plasma protein that inactivates proteinases from all 4 mechanistic classes: serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases (Borth et al., FASEB J, 1992,6: 3345; Baker et al., J Cell Sci, 2002, 115:3719). Another important function of this protein is to serve as a reservoir for cytokines and growth factors, examples of which include TGF, PDGF, and FGF (Asplin et al, Blood, 2001, 97: 3450; Huang et al, J Biol Chem, 1988; 263: 1535). In chronic wounds like diabetic ulcers or venous ulcers, the presence of high amount of proteases leads to rapid degradation of growth factors and delays in wound healing. Thus, a placental membrane wound dressing comprising α2-macroglobulin would constitute an advance in the art.

An in vitro cell migration assay is important for assessing the wound healing potential of a skin substitute. The process of wound healing is highly complex and involves a series of structured events controlled by growth factors (Goldman, Adv Skin Wound Care, 2004, 1:24). These events include increased vascularization, infiltration by inflammatory immune cells, and increases in cell proliferation. The beginning stages of wound healing revolve around the ability of individual cells to polarize towards the wound and migrate into the wounded area in order to close the wound area and rebuild the surrounding tissue. An assay capable of evaluating the wound healing potential of skin substitutes by examining the correlation between cell migration and wound healing would represent an advance in the art.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutically acceptable placental product.

A placental product according to the present invention comprises an immunocompatible amniotic membrane in a cryopreservation medium (optionally cryopreserved) and viable native therapeutic cells and native therapeutic factors.

In some embodiments, the amniotic membrane of the placental product is selectively devitalized.

There is now provided a placental product that is selectively depleted of substantially all immunogenic cells.

There is now provided a placental product that does not contain ex vivo cultured cells.

In some embodiments, the placental product further comprises a chorionic membrane that is selectively devitalized.

There is now provided a placental product that comprises at least one of Epidermal Growth Factor, IGFBP1, and Adiponectin.

Optionally, the therapeutic factors include one or more of IGFBP1, adiponectin, α2-macroglobulin, bFGF, and EGF. Optionally, the therapeutic factors include MMP-9 and TIMP1, wherein the ratio of MMP-9:TIMP1 is from about 7 to about 10. Optionally, the therapeutic factors include IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally, the therapeutic factors include IGFBP1, adiponectin, α2-macroglobulin, bFGF, MMP-9, and TIMP1, wherein the the ratio of MMP-9:TIMP1 is from about 7 to about 10 to one. Optionally, the therapeutic factor is present in a substantial amount in comparison to unprocessed human placental membrane. Optionally, each placental product embodiment optionally is devoid of ex-vivo expanded cultured cells.

The present invention also provides a method of manufacturing a placental product comprising: obtaining a placenta, wherein the placenta comprises an amniotic membrane, selectively depleted of immunogenicity, and cryopreserving the placenta, thereby providing a placental product. According to the present invention, the step of selective depletion comprises removing immunogenic cells (e.g. CD14+ macrophages and vascularized tissue-derived cells) and/or immunogenic factors (e.g. TNFα). Optionally, the step of selective depletion comprises removing CD14+ macrophages by refrigerating the placental product for a period of time (e.g. about 30-60 mins.) at a temperature above freezing (e.g. at 2-8° C.), and then freezing, whereby CD14+ macrophages are selectively killed relative to therapeutic cells.

The present invention also provides a method of screening a placental product for therapy comprising assaying the placental product for immunogenicity and/or therapeutic value. Optionally, the step of assaying the placental product for immunogenicity comprises a Mixed Lymphocyte Reaction (MLR) and/or Lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-α secretion. Optionally, the step of assaying the placental product for therapeutic value comprises assaying the placental product for cell migration induction.

The present invention also provides a method of treating a subject comprising administering a placental product to the subject. Optionally, the step of administering comprises applying the placental product to a wound, for example, topically applying the placental product to a skin wound. In one embodiment, a placental product is used in a tendon or ligament surgery to promote healing of a tendon or ligament.

The present inventors have identified a need for the development of amniotic membrane products comprising at least one of IGFBP1, and adiponectin, providing superior wound healing properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts freezing rates of various freezing methods.

FIG. 2 depicts process cell recovery for amniotic membrane as a function of cryo volume.

FIG. 3 depicts process cell recovery for chorionic membrane as a function of cryo volume.

FIG. 4 depicts the effects of refrigeration time and freezing parameters on process (cryopreservation) cell recovery for the amniotic membrane.

FIG. 5 depicts the effects of refrigeration time and freezing parameters on process (cryopreservation) cell recovery for the chorionic membrane.

FIG. 6 shows representative images of the live/dead staining of the epithelial layer of fresh amniotic membrane.

FIG. 7 depicts expression of IL-2sR from T-cells stimulated by placental derived cells from various membrane preparations.

FIG. 8 depicts expression of IL-2sR from T-cells stimulated by placental derived cells from various membrane preparations after cryopreservation.

FIG. 9 depicts LPS stimulated TNF a released from various membrane preparations.

FIG. 10 shows images of cultured cells isolated from various membrane preparations.

FIG. 11 depicts a correlation between IL-2sR release in an MLR assay of various membrane preparations and the number of CD45+ cells.

FIG. 12 depicts the IL-2sR release in an MLR assay of amniotic membrane preparations and the number of CD45+ cells.

FIG. 13 depicts expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic and/or chorionic membranes.

FIG. 14 depicts expression of IFN-2α and TGF-β3 in amniotic membrane homogenates.

FIG. 15 depicts expression of BMP-2, BMP-4, PLAB, PIGF (A), and IGF-1 (B) in amniotic membrane homogenates.

FIG. 16 depicts the ratio of MMPs to TIMPs in various membrane products.

FIG. 17 depicts expression of EGF in chorion and amnion membranes measured by ELISA in two separate placenta donors.

FIG. 18 depicts the Cell Biolabs 24-well Cytoselect wound healing assay.

FIG. 19 depicts representative images of HMVECs treated with 5% conditioned media from amniotic, chorionic, or a combination of amniotic/chorionic preparations.

FIG. 20 depicts the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 1.

FIG. 21 depicts the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 2.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following definitions apply:

“Examplary” (or “e.g.” or “by example”) means a non-limiting example.

“hCMSCs: means human chorionic membrane stromal cells . hCMSCs are generally positive for CD73?, CD70, CD90, CD105, and CD166; negative for CD45 and CD34,. hCMSCs differentiate to mesodermal lineages (osteogenic, chondrogenic, and adipogenic).

“Selective depletion of immunogenicity” or “selective depletion of immunogenic cells or factors” or “selective depletion” means a placental product that retains live therapeutic cells and/or retains therapeutic efficacy for the treatment of tissue injury yet is free, substantially free, or depleted of at least one of immune cell type (e.g. CD14+ macrophages, trophoblasts, and/or vascular-tissue derived cells) and/or immunogenic factor that are otherwise present in a native placenta or amniotic membrane.

“MSC” means mesenchymal stem cells and include fetal, neonatal, adult, or post-natal. “MSCs” include amniotic MSCs (AMSCs). MSCs generally express one or more of CD73, CD90, CD105, and CD166.

“Native cells” means cells that are native, resident, or endogenous to the placental membrane, i.e. cells that are not exogenously added to the placental membrane.

“Native factors” means placental membrane factors that are native, resident, or endogenous to the placental membrane, i.e. factors that are not exogenously added to the placental membrane.

“Placental products” means the instant placental products disclosed and claimed herein.

“Substantial amount” of an element of the present invention, e.g. native factors, native cells, therapeutic factors, or selective depletion, means a value at least about 10% or more in comparison to an unprocessed, not cryopreserved, fresh membrane sample. A substantial amount can optionally be at least about 50%.

“Therapeutic cells” or “beneficial cells” means stromal cells, MSCs, fibroblasts, and/or epithelial cells.

“Therapeutic factors” means placenta- or amniotic membrane- derived factors that promote wound healing. Examples include IGFBP1, adiponectin, α2-macroglobulin, and/or bFGF. Other examples include MMP-9 and TIMP1.

“Stromal cells” refers to a mixed population of cells present (optionally in native proportions) composed of neonatal mesenchymal stem cells and neonatal fibroblasts. Both neonatal mesenchymal stem cells and neonatal fibroblasts are immunoprivileged; neither express surface proteins present on immunogenic cell types.

In some embodiments, the present technology discloses placental products for clinical use, including use in wound healing such as diabetic foot ulcers, venous leg ulcers, and burns. The manufacturing process optionally eliminates essentially all potentially immunogenic cells from the placental membrane while preserving of specific cells that play an important role in wound healing.

In some embodiments, the present technology discloses a placental product that is selectively devitalized. There is now provided a placental product that is selectively depleted of substantially all immunogenic cells. There is now provided a placental product that does not contain ex vivo cultured cells. There is now provided a placental product that comprises at least one of IGFBP1, and adiponectin. There is now provided a placental product that comprises Epidermal Growth Factor, IGFBP1. There is now provided a placental product that comprises adiponectin.

In some embodiments, the present technology discloses a method of cyropreserving a placental product that preserves the viability of specific beneficial cells that are the primary source of factors for the promotion of healing to the wound healing process while selectively depleting immunogenic cells (e.g. killing or rendering non-immunogenic).

In some embodiments, the present technology discloses a bioassay to test immunogenicity of manufactured placental products.

In some embodiments, the present technology discloses a placental product exhibiting a therapeutic ratio of MMP to TIMP comparable to that exhibited in vivo. The present inventors have identified a need for the development of placental products exhibiting a ratio of MMP-9 and TIMP1 of about 7-10 to one.

In some embodiments, the present technology discloses a placental product that comprises α2-macroglobulin.

There is now provided a placental product that inactivates substantially all serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases present in the amniotic membrane. There is now provided a placental product that inactivates substantially all serine proteinases present in the amniotic membrane. There is now provided a placental product that inactivates substantially all cysteine proteinases present in the amniotic membrane. There is now provided a placental product that inactivates substantially all aspartic proteinases present in the amniotic membrane. There is now provided a placental product that inactivates substantially all metalloproteinases present in the amniotic membrane.

In some embodiments, the present technology discloses a placental product that comprises bFGF.

In some embodiments, the present technology discloses a placental product exhibiting a protease-to-protease inhibitor ratio favorable for wound healing.

In some embodiments, the present technology discloses a cell migration assay capable of evaluating the wound-healing potential of a placental product.

IGFBP1 and adiponectin are among the factors that are important for wound healing. Evaluation of proteins derived from placental products prepared according to the presently disclosed technology reveal that EGF is one of the major factors secreted in higher quantities by these tissues. Additionally, the importance of EGF for wound healing together with high levels of EGF detected in the presently disclosed amniotic membranes support selection of EGF as a potency marker for evaluation of membrane products manufactured for clinical use pursuant to the present disclosure.

The present technology discloses a cryopreservation procedure for a placental product that selectively depletes immunogenic cells and preserves the viability of other beneficial cells (including at least one or 2 or all of mesenchymal stem cells, epithelial cells and fibroblasts . In some embodiments, the beneficial cells are the primary source of factors for the promotion of healing.

Placental products, their usefulness, and their immunocompatability are surprisingly enhanced by depletion of maternal trophoblast and selective elimination of CD14+ fetal macrophages. Immunocompatability can be demonstrated by any means commonly known by the skilled artisan, such demonstration can be performed by the mixed Lymphocyte Reaction (MLR) and by lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-α secretion.

The instant placental products contain bFGF optionally at a substantial concentration.

The instant placental products optionally secrete factors that stimulate cell migration and/or wound healing. The presence of such factors can be demonstrated by any commonly recognized method,

For example, commercially available wound healing assays exist (Cell Biolabs) and cell migration can be assessed by cell line HMVEC (Lonza Inc.). In one embodiment, conditioned medium from the present placental products enhance cell migration.

The placental products disclosed herein are useful in treating a number of wounds including: tendon repair, cartilage repair (e.g. femoral condyle, tibial plateau), ACL replacement at the tunnel/bone interface, dental tissue augmentation, fistulas (e.g. Crohn's disease, G-tube, tracheoesophogeal), missing tissue at adhesion barriers (e.g. nasal septum repair, vaginal wall repair, abdominal wall repair, tumor resection), dermal wounds (e.g. partial thickness burns, toxic epidermal necrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g. diabetic ulcers (e.g. foot), venous leg ulcers), surgical wounds, hernia repair, tendon repair, bladder repair, periosteum replacement, keloids, organ lacerations, epithelial defects, and repair or replacement of a tympanic membrane.

The placental products disclosed herein exhibit one or more of the following properties beneficial to the wound healing process:

-   a. an epithelial cell layer, wherein the approximate number of cells     per cm2 of the amniotic membrane is about 10,000 to about 360,000 or     about 40,000 to about 90,000. -   b. a thick basement membrane (comprising one or more of Collagen     Type I, Ill, IV, laminin, and fibronectin), -   c. a stromal cell layer; -   d. an amniotic membrane with a thickness of about 20 to about 50 μm, -   e. high thrombin activity, -   f. low immunogenicity, -   g. cryopreserved/cryopreserveable, -   h. amniotic MSC, -   i. analgesic effect -   j. reduces scarring, -   k. anti-inflammatory proteins such as IL-1a and IL-10, -   l. anti-inflammatory suppression of lymphocyte reactivity in vitro,     for example by inhibition of CD8+ and CD4+ proliferation or     increased CD4+ Treg cells, -   m. antibacterial proteins such as defensins and allantoin     (bacteriolytic proteins), -   n. angiogenic and mitogenic factors that promote     re-epithelialization such as EGF, HGF, and VEGF, -   o. cells that are positive for CD70, CD90, CD105, and CD166 and     negative for CD45 and CD34, -   p. cells that express HLA-G, -   q. cells that express a placenta specific MHC-1 antigen important to     immune tolerance that inhibits both NK cytolysis and and     T-cell-mediated cytolysis and activation of immune cells (e.g.     interferon-y secretion);, -   r. cells that express IDO and FAS ligand, which likely contribute to     immune tolerance, -   s. cells with a capacity to differentiate into 1-Human Amniotic     Epithelial Cells (hAECs) -   t. cells with a capacity to differentiate to neural, hepatocyte, and     pancreatic cells, -   u. Human Amniotic Membrane Stromal Cells (hAMSCs) differentiate to     mesodermal lineages (osteogenic, chondrogenic, and adipogenic) and     to all three germ layers-ectoderm (neural), mesoderm (skeletal     muscle, cardiomyocytic, and endothelial), and ectoderm (pancreatic), -   v. Cells that expression CD49d by hAMSCs distinguishes hAMSCs from     hAECs, -   w. hAMSCs that positive for the embryonic cytoplasmic marker Oct-4     that plays a role in maintaining pluripotency and self renewal, -   x. hAECs that are positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,     and negative for SSEA-4 and non-tumorogenic.

The present inventors have now identified a need for the development of placental products that do not contain ex vivo cultured cells.

The present inventors have now identified a need for the development of placental products comprising IGFBP1.

The present inventors have now identified a need for the development of placental products comprising adiponectin.

The present inventors have now identified a need for the development of placental products exhibiting a protease-to-protease inhibitor ratio favorable for wound healing.

The present inventors have now identified a need for the development of a method of cyropreserving placental products that preserves the viability of specific cells that are other beneficial cells that are the primary source of factors for the promotion of healing to the wound healing process while selectively depleting immunogenic cells from chorionic membranes.

The present inventors have now identified a need for the development of a bioassay to test immunogenicity of manufactured placental products.

The present inventors have now identified a need for the development of placental products exhibiting a ratio of MMP to TIMP comparable to that exhibited in vivo. The present inventors have now identified a need for the development of placental products exhibiting a ratio of MMP-9 and TIMP1 of about 7 - 10 to one.

The present inventors have now identified a need for the development of placental products that comprise α2-macroglobulin.

The present inventors have now identified a need for the development of placental products that inactivate serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases. The present inventors have now identified a need for the development of placental products that inactivate serine proteinases. The present inventors have now identified a need for the development of placental products that inactivate cysteine proteinases. The present inventors have now identified a need for the development of placental products that inactivate aspartic proteinases. The present inventors have now identified a need for the development of placental products that inactivate metalloproteinases.

The present inventors have now identified a need for the development of placental products that comprise bFGF.

The present inventors have now identified a need for the development of a cell migration assay to evaluate the potential of placental membrane products.

The present inventors have now identified a need for the development of a placental product for wound healing that comprises mesenchymal stem cells, epithelial cells and fibroblasts.

Placental Product

Overview

One embodiment of the present invention provides a placental product comprising a cryopreservation medium and an amniotic membrane, wherein the amniotic membrane comprises viable native therapeutic cells and native therapeutic factors, and wherein the cryopreservation medium comprises a cryopreserving amount of a cryopreservative. According to this embodiment, the amniotic membrane is substantially free of at least one at least one or both of the following immunogenic cell types: CD14+ macrophages and vascularized tissue-derived cells.

In one embodiment, the amniotic membrane comprises one or more layers which exhibit the architecture of the native amniotic membrane (e.g. has not been homogenized or treated with collagenase).

In one embodiment, the placental product is suitable for dermal application to a wound.

With the teachings provided herein, the skilled artisan can now produce the present placental products. The present disclosure provides methods of manufacture that produce the technical features of the present placental products. Accordingly, in one embodiment, the placental product is manufactured by steps taught herein. The present placental products are not limited to products manufactured by the methods taught here. For example, products of the present invention could be produced through methods that rely on screening steps; e.g. steps to screen for preparations with the described technical features and superior properties.

The present placental product comprises one or more of the following technical features:

-   a. the viable therapeutic native cells are capable of     differentiating into cells of more than one lineage (e.g.     osteogenic, adipogenic and/or chonodrogenic lineages), -   b. the native therapeutic factors include IGFBP1, -   c. the native therapeutic factors include adiponectin, -   d. the native therapeutic factors include α2-macroglobulin, -   e. the native therapeutic factors include bFGF, -   f. the native therapeutic factors include EGF, -   g. the native therapeutic factors include MMP-9 and TIMP1, -   h. the native therapeutic factors include MMP-9 and TIMP1 in a ratio     of about 7 to about 10, -   i. the placental product does not comprise ex-vivo cultured cells, -   j. the cryopreservative medium is present in an amount of greater     than about 20 ml or greater than about 50 ml, -   k. the cryopreservative comprises DMSO, -   l. cryopreservative comprises DMSO in a majority amount, -   m. the cryopreservation medium further comprises albumin, optionally     wherein the albumin is HSA, -   n. the cryopreservative comprises DMSO and albumin (e.g. HSA), -   o. comprises about 5,000 to about 240,000 cells/cm² or about 20,000     to about 60,000, -   p. the amniotic membrane comprises at least: about 2,000, or about     2,400, or about 4,000 or about 6,000, or about 8,000, or about     10,000, or about 10,585, or about 15,000 stromal cells per unit cm²     of the amniotic membrane, -   q. the amniotic membrane comprises about 2,000 to about 15,000 of     stromal cells per cm² of the amniotic membrane, -   r. comprises stromal cells wherein at least: about 40%, or about     50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or     about 90%, or about 92.5% of the stromal cells are viable after a     freeze-thaw cycle, -   s. comprises stromal cells wherein about 40% to about 92.5% of the     stromal cells are viable after a freeze-thaw cycle, -   t. the amniotic membrane has a thickness of about 20 μm to about 50     μm, -   u. secretes less than about any of: 420 μg/mL, 350 μg/mL, or 280     μg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm     piece of the tissue product in a tissue culture medium and exposing     the tissue product to a bacterial lipopolysaccharide for about 20 to     about 24 hours, -   v. cryopreservation and thawing, secretes less than about any of:     420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culture     medium upon placing a 2 cm×2 cm piece of the tissue product in a     tissue culture medium and exposing the tissue product to a bacterial     lipopolysaccharide for about 20 to about 24 hours, -   w. after refrigeration, cryopreservation and thawing, secretes less     than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a     tissue culture medium upon placing a 2 cm×2 cm piece of the tissue     product in a tissue culture medium and exposing the tissue product     to a bacterial lipopolysaccharide for about 20 to about 24 hours, -   x. further comprises an chorionic membrane, -   y. amniotic membrane comprises a layer of amniotic epithelial cells, -   z. further comprises an chorionic membrane, wherein the amniotic     membrane and the chorionic membrane are associated to one another in     the native configuration, -   aa. further comprises an chorionic membrane, wherein the amniotic     membrane and the chorionic membrane are not attached to one another     in the native configuration, -   bb. further comprises a chorionic membranewherein the chorionic     membrane comprises about 2 to about 4 times more stromal cells     relative to the amniotic membrane, -   cc. does not comprise a chorionic membrane; -   dd. comprises chorionic membrane, wherein the chorionic membrane     comprises about 2 to about 4 times more stromal cells relative to     the amniotic membrane, and -   ee. is suitable for dermal application to a wound;

Cells

According to the present invention, a placental product comprises native therapeutic cells of the amniotic membrane. The cells comprise one or more of stromal cells, MSCs, fibroblasts, and epithelial cells.

In one embodiment, the native therapeutic cells comprise viable stromal cells.

In one embodiment, the native therapeutic cells comprise viable MSCs.

In one embodiment, the native therapeutic cells comprise viable fibroblasts.

In one embodiment, the native therapeutic cells comprise viable epithelial cells.

In one embodiment, the native therapeutic cells comprise viable MSCs and viable fibroblasts.

In one embodiment, the native therapeutic cells comprise viable MSCs, viable fibroblasts, and viable epithelial cells.

In one embodiment, the native therapeutic cells comprise viable stromal cells and viable epithelial cells.

In one embodiment, the therapeutic native cells are viable cells capable of differentiating into cells of more than one lineage (e.g. osteogenic, adipogenic and/or chonodrogenic lineages).

In one embodiment, the placental product comprises about 10,000 to about 360,000 or about 40,000 to about 90,000 cells per cm².

In one embodiment, the placental product comprises at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 stromal cells per unit cm² of the placental product.

In one embodiment, the placental product comprises about 2,000 to about 15,000 of stromal cells per cm² of the placental product.

In one embodiment, the placental product comprises stromal cells wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable after a freeze-thaw cycle.

In one embodiment, the placental product comprises stromal cells wherein about 40% to about 92.5% of the stromal cells are viable after a freeze-thaw cycle.

In one embodiment, the placental product comprises less than about 1% of CD14+ macrophages per total cells.

In one embodiment, the amniotic membrane of the placental product comprises about 2 to about 4 times less stromal cells relative to a chorionic membrane of the same area derived from the same placenta.

In one embodiment, the placental product further comprises membrane chorionic membrane containing about 2 to about 4 times more stromal cells relative to the amniotic membrane.

In one embodiment, the amniotic membrane of the placental product comprises MSCs in an amount of: at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, about 1% to about 10%, or about 3% to about 10%, relative to the total number of cells in the amniotic membrane of the placental product. Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the MSCs are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises fibroblasts in an amount of: about 1%, about 20% , about 5% to about 15%, at least about 1%, at least about 2%, at least about 3%, or at least about 4 relative to the total number of cells in the amniotic membrane of the placental product. Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the fibroblasts are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises stromal cells in an amount of: about 5% to about 40%, about 5% to about 30%, about 10% to about 30%, about 15% to about 25%, at least about 5%, at least about 10%, or at least about 15%, relative to the total number of cells in the amniotic membrane of the placental product. Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the stromal cells are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises epithelial cells in an amount of: about 60% to about 90%, about 70% to about 90%, about 40% to about 90%, about 50% to about 90%, at least about 40%, at least about 50%, least about 60%, or at least about 70%, relative to the total number of cells in the amniotic membrane of the placental product. Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the epithelial cells are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises MSCs and functional macrophages in a ratio of greater than about any of: 5:1, 7:1, or 10:1.

In one embodiment, the amniotic membrane of the placental product comprises fibroblasts and functional macrophages in a ratio of greater than about any of: 10:1, 15:1, 20:1, or 25:1.

In one embodiment, the amniotic membrane of the placental product comprises fibroblasts and MSCs in a ratio of: about 4:1 to about 1:1 or about 3:1 to about 3:2, or about 2:1.

In one embodiment, the amniotic membrane of the placental product comprises MSCs in an amount of: at least about 1,000 cells/cm²′ at least about 2,000 cells/cm², about 1,000 to about 5,000 cells/cm², or about 2,000 to about 5,000 cells/cm². Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the MSCs are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises fibroblasts in an amount of: at least about 2,000 cells/cm², at least about 4,000 cells/cm², about 2,000 to about 9,000 cells/cm² , or about 2,000 to about 9,000 cells/cm². Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the fibroblasts are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises stromal cells in an amount of: at least about 4,000, at least about 8,000 cells/cm², about 4,000 to about 18,000 cells/cm², or about 4,000 to about 18,000 cells/cm². Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the stromal cells are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises epithelial cells in an amount of: at least about 10,000 cells/cm², at least about 20,000 cells/cm², at least about 32,000 cells/cm², about 10,000 to about 72,000 cells/cm² , about 20,000 to about 72,000 cells/cm² , or about 32,000 to about 72,000 cells/cm² Optionally, at least: about 40%, about 50%, about 60%, or about 70% of the epithelial cells are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental product comprises functional macrophages in an amount of: less than about 3,000 cells/cm², less than about 1,000 cells/cm², or less than about 500 cells/cm².

In one embodiment, the placental product comprises a layer of amniotic epithelial cells.

In one embodiment, the placental product comprises a chorionic membrane but is substantially free of trophoblasts.

In one embodiment, the placental product is substantially free of functional CD14+ macrophages.

In one embodiment, the placental product is substantially free of vascularized tissue-derived cells.

In one embodiment, the placental product is substantially free of trophoblasts, functional CD14+ macrophages, and vascularized tissue-derived cells. Optionally, the placental product comprises viable stromal cells. Optionally, the placental product comprises viable MSCs. Optionally, the placental product comprises viable fibroblasts. Optionally, the placental product comprises viable epithelial cells. Optionally, the placental product comprises viable MSCs, fibroblasts, and epithelial cells.

In one embodiment, the placental product comprises a chorionic membrane but is substantially free of maternal decidual cells.

In one embodiment, the placental product comprises a chorionic membrane but is substantially free of maternal leukocytes and/or trophoblast cells.

In one embodiment, the placental product is substantially free of ex-vivo cultured cells.

Placental Factors

According to the present invention, a placental product comprises native therapeutic factors of the amniotic membrane.

In one embodiment, the factors include one or more of: IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally, the factors are present in amounts/cm² that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally, the factors are present in ratios that are substantially similar to that of a native amniotic membrane or layer thereof. Optionally, the factors are present in amounts/cm² that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include MMP-9 and TIMP1 in a ratio of about 7 to about 10 (e.g about 7). Optionally, the factors are present in amounts/cm² that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include one or more (e.g. a majority or all) of the factors listed in Table 16. Optionally, the factors are present in ratios that are substantially similar to that of a native amniotic membrane or layer thereof. Optionally, the factors are present in amounts/cm² that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the placental product or layer thereof comprises substantially less TNF-α/cm² than a native amniotic membrane or layer thereof, respectively.

In one embodiment, the placental product or layer thereof secretes substantially less TNF-α/cm² than a native placental product or layer thereof, respectively.

In one embodiment, the placental product secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.

In one embodiment, after cryopreservation and thawing, the placental product secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours;

In one embodiment, after refrigeration, cryopreservation and thawing, the placental product secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.

In one embodiment, the placental product further comprises an exogenously added inhibitor of TNF-α. Optionally, the inhibitor of TNF-α is IL-10.

In one embodiment, the product has been treated with an antibiotic

Architecture

A placental product of the present invention comprises one or more layers which exhibit the architecture of the native amniotic membrane. With the teachings provided herein, the skilled artisan will recognize placental layers that exhibit native architecture, for example, layers that have not been homogenized or treated with collagenase or other enzyme that substantially disrupts the layer.

In one embodiment, the placental product comprises a stromal cell layer with native architecture of the amniotic membrane.

In one embodiment, the placental product comprises a basement membrane with native architecture of the amniotic membrane.

In one embodiment, the placental product comprises an epithelial cell layer with native architecture of the amniotic membrane.

In one embodiment, the placental product comprises a stromal cell layer and a basement layer with native architecture of the amniotic membrane.

In one embodiment, the placental product comprises a stromal layer, a basement layer, and an epithelial cell layer with native architecture of the amniotic membrane.

In one embodiment, the placental product or amniotic membrane thereof has a thickness of about 20 μm to about 50 μm.

In one embodiment, the placental product comprises a chorionic membrane but is substantially free of trophoblasts. In one embodiment, the placental product comprises a basement membrane with native architecture of the chorionic membrane and the chorionic membrane is substantially free of trophoblasts. Optionally, the maternal side of the chorionic membrane comprises fragments of extracellular matrix proteins in a concentration substantially greater than that of a native chorionic membrane. Optionally, the placental product has been treated with Dispase (e.g. Dispase II) and/or a substantial portion of the extracellular matrix protein fragments comprises terminal leucine or phenylalanine.

In one embodiment, the placental product further comprises a chorionic membrane. Optionally, the amniotic membrane and the chorionic membrane in the placental product are associated to one another in the native configuration. Alternatively, the amniotic membrane and the chorionic membrane are not attached to one another in the native configuration.

In one embodiment, the placental product does not comprise a chorionic membrane.

Formulation

According to the present invention, the placental product can be formulated with a cryopreservation medium.

In one embodiment, the cryopreservation medium comprising one or more cell-permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof.

Optionally, the cryopreservation medium comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof.

Optionally, the cryopreservation medium comprises one or more non cell-permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.

Other examples of useful cryopreservatives are described in “Cryopreservation” (BioFiles Volume 5 Number 4—Sigma-Aldrich® datasheet).

In one embodiment, the cryopreservation medium comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises DMSO. Optionally, the cryopreservation medium does not comprise glycerol in a majority amount. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises additional components such as albumin (e.g. HSA or BSA), an electrolyte solution (e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 15% albumin by weight and about 5% to about 20% cryopreservative by volume (e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g. in a majority amount).

In one embodiment, the placental product is formulated in greater than about 20 ml or greater than about 50 ml of cryopreservation medium. Optionally, the cryopreservative comprises DMSO (e.g. in a majority amount). Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

In one embodiment, the placental product is placed on nitrocellulose paper.

In one embodiment, the placenta is cut into a plurality of sections. Optionally, the sections are less than about 10 cm×10 cm. Optionally, the sections are between about 2 cm×2 cm and 5 cm×5 cm.

Manufacture

Overview

A placental product of the present invention can manufactured from a placenta in any suitable manner that provides the technical features taught herein. According to the present invention, a placenta product comprises at least an immunocompatible amniotic membrane.

In one embodiment, a placental product is manufactured by a method comprising:

-   a. obtaining a placenta, -   b. selectively depleting the placenta of immunogenicity; and -   c. cryopreserving the placenta.

Optionally, the method comprises a step of removing vascularized tissue from the placenta, for example, by lysing red blood cells, by removing blood clots, or a combination thereof.

Optionally, the method comprises a step of treating the placenta with one or more antibiotics.

Optionally, the method comprises a step of selectively depleting CD14+ macrophages , optionally as demonstrated by a substantial decrease in LPS stimulation of TNFα release.

Optionally, the step of cryopreserving the placenta comprises freezing the placenta in a cryopreservation medium which comprises one or more cell-permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof.

Optionally, the step of cryopreserving the placenta comprises refrigerating for a period of time and then freezing, thereby selectively depleting CD14+ macrophages optionally as demonstrated by a substantial decrease in LPS stimulation of TNFα release.

Optionally, the method comprises retaining a layer of epithelial cells of the amniotic membrane.

Optionally, the method comprises a step of removing the chorionic membrane or portion thereof. Optionally, the method comprises removing trophoblasts from the chorionic membrane while retaining the stromal cell layer, reticular layer, and/or basement membrane of the chorionic membrane.

An examplary placental product of the present invention can be manufactured or provided with a bandage or wound dressing. lmmunocompatability and Selective Depletion

In one embodiment, the invention the placental product is immunocompatible. Immunocompatability can be accomplished by any selective depletion step that removes immunogenic cells or factors or immunogenicity from the placenta (or amniotic membrane thereof).

In one embodiment, the placental product is made immunocompatible by selectively depleting it of functional immunogenic cells. A placenta can be made immunocompatible by selectively removing immunogenic cells from the placenta (or amniotic membrane thereof) relative to therapeutic cells. For example, immunogenic cells can be removed by killing the immunogenic cells or by purification of the placenta there from.

In one embodiment, the placental product is made immunocompatible by selectively depleting trophoblasts, for example, by removal of the trophoblast layer.

In one embodiment, the placenta is made immunocompatible by selective depletion of functional CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFα release or by MLR assay.

In one embodiment, the placenta is made immunocompatible by selective depletion of vascularized tissue-derived cells.

In one embodiment, the placenta is made immunocompatible by selective depletion of functional CD14+ macrophages, trophoblasts, and vascularized tissue-derived cells.

In one embodiment, the placenta product is made immunocompatible by selective depletion of trophoblasts and/or CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFα release or by MLR assay.

Trophoblast Removal

In one embodiment, Immunocompatability (or selective depletion) is accomplished by removal or depletion of trophoblasts from the placental product. Trophoblasts can be removed by removing the chorionic membrane from the placental product or by removing trophoblasts from the chorionic membrane while retaining at least one of the basement layer, reticular layer, or stromal cell layer of the chorionic membrane. Surprisingly, such a placental product has one or more of the following superior features:

-   a. is substantially non-immunogenic; -   b. provides remarkable healing time; and -   c. provides enhanced therapeutic efficacy.

In one embodiment, trophoblasts are removed while retaining the basement layer, reticular layer, and/or stromal cell layer of the chorionic membrane.

Trophoblasts can be removed in any suitable manner which substantially diminishes the trophoblast content of the placental product. Optionally, the trophoblasts are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the chorionic membrane (e.g. MSCs, placental factors, etc). Optionally, a majority (e.g. substantially all) of the trophoblasts are removed.

One method of removing trophoblasts comprises treating the placenta (e.g. chorion or amnio-chorion) with a digestive enzyme such as dispase (e.g. dispase II) and separating the trophoblasts from the placenta. Optionally, the step of separating comprises mechanical separation such as peeling or scraping. Optionally, scraping comprises scraping with a soft instrument such as a finger.

One method of removing trophoblasts comprises treating the chorionic membrane with dispase for about 30 to about 45 minutes separating the trophoblasts from the placenta. Optionally, the dispase is provided in a solution of about less than about 1% (e.g. about 0.5%). Optionally, the step of separating comprises mechanical separation such as peeling or scraping. Optionally, scraping comprises scraping with a soft instrument such as a finger.

Useful methods of removing trophoblasts from a placenta (e.g. chorion) are described by Portmann-Lanz et al. (“Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration”; American Journal of Obstetrics and Gynecology (2006) 194, 664-73), (“Isolation and characterization of mesenchymal cells from human fetal membranes”; Journal Of Tissue Engineering And Regenerative Medicine 2007; 1: 296-305.), and (Concise Review: Isolation and Characterization of Cells from Human Term Placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells“).

In one embodiment, trophoblasts are removed before cryopreservation.

Macrophage Removal

In one embodiment, functional macrophages are depleted or removed from the placental product. Surprisingly, such a placental product has one or more of the following superior features:

-   a. is substantially non-immunogenic; -   b. provides remarkable healing time; and -   c. provides enhanced therapeutic efficacy.

Functional macrophages can be removed in any suitable manner which substantially diminishes the macrophage content of the placental product. Optionally, the macrophages are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g. MSCs, placental factors, etc). Optionally, a majority (e.g. substantially all) of the macrophages are removed.

One method of removing immune cells such as macrophages comprises killing the immune cells by rapid freezing rates such as 60-100° C./min.

Although immune cells can be eliminated by rapid freezing rates, such a method can also be detrimental to therapeutic cells such as stromal cells (e.g. MSCs). The present inventors have discovered a method of selectively killing CD14+ macrophages can be selectively killed by refrigerating the placenta for a period of time (e.g. for at least about 10 min such as for about 30-60 mins) at a temperature above freezing (e.g. incubating at 2-8° C.) and then freezing the placenta (e.g. incubating at −80° C.±5° C.). Optionally, the step of freezing comprises freezing at a rate of less than 10°/min (e.g. less than about 5°/min such as at about 1°/min).

In one embodiment, the step of refrigerating comprises soaking the placenta in a cryopreservation medium (e.g. containing DMSO) for a period of time sufficient to allow the cryopreservation medium to penetrate (e.g. equilibrate with) the placental tissues. Optionally, the step of freezing comprises reducing the temperature at a rate of about 1°/min. Optionally, the step of freezing comprises freezing at a rate of less than 10°/min (e.g. less than about 5°/min such as at about 1°/min).

In one embodiment, the step of refrigerating comprises soaking the placenta in a cryopreservation medium (e.g. containing DMSO) at a temperature of about -10-15° C. (e.g. at 2-8° C.) for at least about any of: 10 min, 20 min, 30 min, 40 min, or 50 min. In another embodiment the step of refrigerating comprises soaking the placenta in a cryopreservation medium (e.g. containing DMSO) at a temperature of about -10-15° C. (e.g. at 2-8° C.) for about any of: 10-120, 20-90 min, or 30-60 min. Optionally, the step of freezing comprises freezing at a rate of less than 10°/min (e.g. less than about 5°/min such as at about 1°/min).

Removal of Vascularized Tissue-Derived Cells

In one embodiment, vascularized tissue-derived cells (or vascularized tissue) are depleted or removed from the placental product. Surprisingly, such a placental product has one or more of the following superior features:

-   a. is substantially non-immunogenic; -   b. provides remarkable healing time; and -   c. provides enhanced therapeutic efficacy.

Vascularized tissue-derived cells can be removed in any suitable manner which substantially diminishes such cell content of the placental product. Optionally, the vascularized tissue-derived cells are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g. MSCs, placental factors, etc).

In one embodiment, removal of vascularized tissue-derived cells comprises rinsing the amniotic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells.

In one embodiment, removal of vascularized tissue-derived cells comprises treating the amniotic membrane with an anticoagulant (e.g. citrate dextrose solution).

In one embodiment, removal of vascularized tissue-derived cells comprises rinsing the amniotic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g. citrate dextrose solution).

In one embodiment, the chorionic membrane is retained and removal of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord. The chorion on the umbilical side of the placenta is not removed due to the vascularization on this side.

In one embodiment, the chorionic membrane is retained and removal of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and rinsing the amniotic membrane and chorionic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells.

In one embodiment, the chorionic membrane is retained and removal of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and treating the amniotic membrane and chorionc membrane with an anticoagulant (e.g. citrate dextrose solution).

In one embodiment, the chorionic membrane is retained and emoval of vascularized tissue-derived cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord, rinsing the chorionic membrane amniotic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g. citrate dextrose solution).

Selective Depletion of Immunogenicity as Demonstrated by a Substantial Decrease in LPS Stimulation of TNFα Release.

In one embodiment, the placental product is selectively depleted of immunogenicity as demonstrated by a reduction in LPS stimulated TNF-α release. In one embodiment, the placental product is selectively depleted of macrophages.

In one embodiment, TNF-α is depleted by killing or removal of macrophages.

In one embodiment, TNF-α is functionally depleted by treatment with IL-10, which suppresses TNF-α secretion.

Preservation

A placental product of the present invention may be used fresh or may be preserved for a period of time. Surprisingly, cryopreservation results in immunocompatible placental products.

In one embodiment, a placental product is cryopreserved. A placental product may be cryopreserved by incubation at freezing temperatures (e.g. a −80° C.±5° C.) in a cryopreservative medium.

Cryopreservation can comprise, for example, incubating the placental product at 4° C. for 30-60 min, and then incubating at −80° C. until use. The placental product may then be thawed for use. Optionally, the placental product is cryopreserved in a manner such that cell viability is retained surprisingly well after a freeze-thaw cycle.

In one embodiment, cryopreservation comprises storage in a cryopreservation medium comprising one or more cell-permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof. Optionally, the cryopreservation medium comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof. Optionally, the cryopreservation medium comprises one or more non cell-permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof. Other examples of useful cryopreservatives are described in “Cryopreservation” (BioFiles Volume 5 Number 4—Sigma-Aldrich® datasheet).

In one embodiment, the cryopreservation medium comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises DMSO. Optionally, the cryopreservation medium does not comprise glycerol in a majority amount. Optionally, the cryopreservation medium does not comprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises additional components such as albumin (e.g. HSA or BSA), an electrolyte solution (e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 15% albumin by weight and about 5% to about 20% cryopreservative by volume (e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g. in a majority amount).

In one embodiment, cryopreservation comprises placing the placenta on nitrocellulose paper.

In one embodiment, the placenta is cut into a plurality of sections before cryopreservation. Optionally, the sections are placed on nitrocellulose paper before refrigeration.

Methods of Use

The placental products of the present invention may be used to treat any tissue injury. A method of treatment may be provided, for example, by administering to a subject in need thereof, a placental product of the present invention.

A typical administration method of the present invention is topical administration. Administering the present invention can optionally involve administration to an internal tissue where access is gained by a surgical procedure.

Placental products can be administered autologously, allogeneically or xenogeneically.

In one embodiment, a present placental product is administered to a subject to treat a wound. Optionally, the wound is a laceration, scrape, thermal or chemical burn, incision, puncture, or wound caused by a projectile. Optionally, the wound is an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wounds, congenital wound, ulcer, or pressure ulcer. Such wounds may be accidental or deliberate, e.g., wounds caused during or as an adjunct to a surgical procedure. Optionally, the wound is closed surgically prior to administration.

In one embodiment, a present placental product is administered to a subject to treat a burn. Optionally, the burn is a first-degree burn, second-degree burn (partial thickness burns), third degree burn (full thickness burns), infection of burn wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed burn, or burn wound impetigo.

In one embodiment, a present placental product is administered to a subject to treat an ulcer, for example, a diabetic ulcer (e.g. foot ulcer).

In one embodiment, a placental product is administered by placing the placental product directly over the skin of the subject, e.g., on the stratum corneum, on the site of the wound, so that the wound is covered, for example, using an adhesive tape. Additionally or alternatively, the placental product may be administered as an implant, e.g., as a subcutaneous implant.

In one embodiment, a placental product is administered to the epidermis to reduce rhtids or other features of aging skin. Such treatment is also usefully combined with so-called cosmetic surgery (e.g. rhinoplasty, rhytidectomy, etc.).

In one embodiment, a placental product is administered to the epidermis to accelerate healing associated with a dermal ablation procedure or a dermal abrasion procedure (e.g. including laser ablation, thermal ablation, electric ablation, deep dermal ablation, sub-dermal ablation, fractional ablation, and microdermal abrasion).

Other pathologies that may be treated with placental products of the present invention include traumatic wounds (e.g. civilian and military wounds), surgical scars and wounds, spinal fusions, spinal cord injury, avascular necrosis, reconstructive surgeries, ablations, and ischemia.

In one embodiment, a placental product of the present invention is used in a tissue graft procedure. Optionally, the placental product is applied to a portion of the graft which is then attached to a biological substrate (e.g. to promote healing and/or attachment to the substrate). By way of non-limiting example, tissues such as skin, cartilage, ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenter and sinew can be used as tissue graft.

In one embodiment, a placental product is used in a tendon or ligament surgery to promote healing of a tendon or ligament. Optionally, the placental product is applied to portion of a tendon or ligament which is attached to a bone. The surgery can be any tendon or ligament surgery, including, e.g. knee surgery, shoulder, leg surgery, arm surgery, elbow surgery, finger surgery, hand surgery, wrist surgery, toe surgery, foot surgery, ankle surgery, and the like. For example, the placental product can be applied to a tendon or ligament in a grafting or reconstruction procedure to promote fixation of the tendon or ligament to a bone.

Through the insight of the inventors, it has surprisingly been discovered that placental products of the present invention provide superior treatment (e.g. healing time and/or healing strength) for tendon and ligament surgeries. Tendon and ligament surgeries can involve the fixation of the tendon or ligament to bone. Without being bound by theory, the present inventors believe that osteogenic and/or chondrogenic potential of MSCs in the present placental products promotes healing process and healing strength of tendons or ligaments. The present inventors believe that the present placental products provide an alternative or adjunctive treatment to periosteum-based therapies. For example, useful periosteum based treatments are described in Chen et al. (“Enveloping the tendon graft with periosteum to enhance tendon-bone healing in a bone tunnel: A biomechanical and histologic study in rabbits”; Arthroscopy. 2003 Mar;19(3):290-6), Chen et al. (“Enveloping of periosteum on the hamstring tendon graft in anterior cruciate ligament reconstruction”; Arthroscopy. 2002 May-June; 18(5):27E), Chang et al. (“Rotator cuff repair with periosteum for enhancing tendon-bone healing: a biomechanical and histological study in rabbits”; Knee Surgery, Sports Traumatology, Arthroscopy Volume 17, Number 12, 1447-1453), each of which are incorporated by reference.

As non-limiting example of a method of tendon or ligament surgery, a tendon is sutured to and/or wrapped or enveloped in a placental membrane and the tendon is attached to a bone. Optionally, the tendon is placed into a bone tunnel before attached to the bone.

In one embodiment, the tendon or ligament surgery is a graft procedure, wherein the placental product is applied to the graft. Optionally, the graft is an allograft, xenograft, or an autologous graft.

In one embodiment, the tendon or ligament surgery is repair of a torn ligament or tendon, wherein the placental product is applied to the torn ligament or tendon.

Non-limiting examples of tendons to which a placental product can be applied include a digitorum extensor tendon, a hamstring tendon, a bicep tendon, an Achilles Tendon, an extensor tendon, and a rotator cuff tendon.

In one embodiment, a placental product of the present invention is used to reduce fibrosis by applying the placental product to a wound site.

In one embodiment, a placental product of the present invention is used as an anti-adhesion wound barrier, wherein the placental product is applied to a wound site, for example, to reduce fibrosis (e.g. postoperative fibrosis).

Non-limiting examples of wound sites to which the placental product can be applied include those that are surgically induced or associated with surgery involving the spine, laminectomy, knee, shoulder, or child birth, trauma related wounds or injuries, cardiovascular procedures, angiogenesis stimulation, brain/neurological procedures, burn and wound care, and ophthalmic procedures. For example, optionally, the wound site is associated with surgery of the spine and the stromal side of the placental product is applied to the dura (e.g. the stromal side facing the dura). Direction for such procedures, including the selection of wound sites and/or methodologies, can be found, for example, in WO 2009/132186 and US 2010/0098743, which are hereby incorporated by reference.

A placental product of the present invention can optionally be used to reduce adhesion or fibrosis of a wound. Postoperative fibrosis is a natural consequence of all surgical wound healing. By example, postoperative peridural adhesion results in tethering, traction, and compression of the thecal sac and nerve roots, which cause a recurrence of hyperesthesia that typically manifests a few months after laminectomy surgery. Repeated surgery for removal of scar tissue is associated with poor outcome and increased risk of injury because of the difficulty of identifying neural structures that are surrounded by scar tissue. Therefore, experimental and clinical studies have primarily focused on preventing the adhesion of scar tissue to the dura matter and nerve roots. Spinal adhesions have been implicated as a major contributing factor in failure of spine surgery. Fibrotic scar tissue can cause compression and tethering of nerve roots, which can be associated with recurrent pain and physical impairment.

Without being bound by theory, the present inventors believe that placental products taught herein are useful to reduce adhesion or fibrosis of a wound, at least in part, because the placental products can perform the very critical function in-situ of providing a immunoprivileged environment (i.e. relatively high resistance against immune responses) in the human development process. One advantage of the wound dressings and processes of the present invention is that an anti-adhesion barrier is provided which can be used to prevent adhesions following surgery, and in particular following back surgery.

In the preceding paragraphs, use of the singular may include the plural except where specifically indicated. As used herein, the words “a,” “an,” and “the” mean “one or more,” unless otherwise specified. In addition, where aspects of the present technology are described with reference to lists of alternatives, the technology includes any individual member or subgroup of the list of alternatives and any combinations of one or more thereof.

The disclosures of all patents and publications, including published patent applications, are hereby incorporated by reference in their entireties to the same extent as if each patent and publication were specifically and individually incorporated by reference.

It is to be understood that the scope of the present technology is not to be limited to the specific embodiments described above. The present technology may be practiced other than as particularly described and still be within the scope of the accompanying claims.

Likewise, the following examples are presented in order to more fully illustrate the present technology. They should in no way be construed, however, as limiting the broad scope of the technology disclosed herein.

The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific embodiments of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

EXAMPLES

Other features and embodiments of the present technology will become apparent from the following examples which are given for illustration of the present technology rather than for limiting its intended scope.

Example 1 Characterization of Placental Membranes

Cells in placental membranes were characterized by Fluorescence Activated Cell Sorting (FACS) demonstrated the presence of stromal cells (Mesenchymal Stem Cell-like cells) in addition to fetal epithelial cells and fibroblasts.

One unique characteristic of the presently disclosed placental products is the presence of MSCs, which have been shown to be one of three types of cells (in addition to epithelial cells and fibroblasts) that are important for wound healing. Placental membranes secrete a variety of factors involved in wound healing such as angiogenic factors, factors supporting proliferation and migration of epithelial cells and fibroblasts, factors attracting endothelial stem cells from blood circulation to the wound site, antibacterial factors, and others.

Evaluation of proteins secreted by examplary placental products of the invention in comparison to Apligraf and Dermagraft demonstrated a number of growth factors present in the tested products that are involved in wound healing. Examples are Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor (TGF) and others. However, several unique factors including Epidermal Growth Factor (EGF), which is one of the key factors for wound healing, are present in placental membranes and absent in Apligraf and Dermagraft. Also, placental membranes have a favorable protease-to-protease inhibitor ratio for wound healing. In an in vitro model of wound healing (cell migration assay, disclosed herein), the present inventors have demonstrated that placental membranes secrete factors promoting cell migration that will support wound closure.

Example 2 Exemplary Manufacturing Process of a Placental Product

In one embodiment, the present invention is a method of manufacturing a placental product comprising an amniotic membrane and optionally a chorionic membrane from placenta post partum. One such method is:

-   a. Remove umbilical cord close to placental surface, -   b. Blunt dissect of the amnion to placental skirt, -   c. Flip placenta over and completely remove amnion, -   d. Rinse amnion in PBS to remove red blood cells, -   e. Rinse amnion once with 11% ACD-A solution to assist in red blood     cell removal, -   f. Rinse amnion with PBS to remove ACD-A solution, -   g. Use PBS to remove any remaining blood from the amnion, -   h. Gently remove the connective tissue layer from the amnion, -   i. Place the amnion in PBS and set aside, -   j. Place the amnion into a bottle containing antibiotic solution and     incubate at 37° C. ±2° C. for 24-28 hrs, -   k. Remove bottle from the incubator and rinse membrane with PBS to     remove antibiotic solution, -   l. Mount amnion (epithelial side up) on reinforced nitrocellulose     paper and cut to size, -   m. Place into an FP-90 cryobag and heat seal, -   n. Add 50 mL cryopreservation solution to the bag through a syringe     and remove any air trapped within the bag with the syringe, -   o. Tube seal the solution line on the FP-90 bag, -   p. Place filled bag into secondary bag and heat seal, -   q. Place unit into packaging carton, -   r. Refrigerate at 2-8° C. for 30-60 minutes, Freeze at −80° C.±5° C.     inside a Styrofoam container.

Example 3 Exemplary Manufacturing Process of a Placental Product Containing an Amniotic Membrane and a Chorionic Membrane

In one embodiment, the present invention is a method of manufacturing a placental product comprising an amniotic membrane and optionally a chorionic membranes from placenta post partum. One such method is:

-   a. Remove umbilical cord close to placental surface, -   b. Blunt dissect of the amnion to placental skirt, -   c. Flip placenta over and completely remove amnion, -   d. Remove chorion by cutting around placental skirt, -   e. Rinse both membranes in PBS to remove red blood cells, -   f. Rinse both membranes once with 11% ACD-A solution to assist in     red blood cell removal, -   g. Rinse both membranes with PBS to remove ACD-A solution, \ -   h. Treat chorion in 0.5% dispase solution at 37° C.±2° C. for 30-45     minutes, optionally, during dispase incubation period, use PBS to     remove any remaining blood from the amnion, -   i. Gently remove the connective tissue layer from the amnion, -   j. Place the amnion in PBS and set aside, -   k. When dispase treatment is complete, rinse chorion with PBS to     remove dispase solution, -   l. Gently remove trophoblast layer from the chorion, -   m. Place the amnion and chorion each into a bottle containing     antibiotic solution and incubate at 37° C.±2° C. for 24-28 hrs, -   n. Remove bottles from the incubator and rinse each membrane with     PBS to remove antibiotic solution, -   o. Mount amnion (epithelial side up) or chorion on reinforced     nitrocellulose paper and cut to size, -   p. Place each piece into an FP-90 cryobag and heat seal, -   q. Add 50 mL cryopreservation solution to the bag through a syringe     and remove any air trapped within the bag with the syringe, -   r. Tube seal the solution line on the FP-90 bag, -   s. Place filled bag into secondary bag and heat seal, -   t. Place unit into packaging carton, -   u. Refrigerate at 2-8° C. for 30-60 minutes, -   v. Freeze at −80° C.±5° C. inside a Styrofoam container.

Example 4 Exemplary Placental Product Manufacturing Process

One method manufacturing a placental product comprising an amniotic membrane according to the presently disclosed manufacturing procedure is as follows:

One method of manufacturing a placental product comprising a chorionic membrane product and an amniotic membrane product according to the presently disclosed manufacturing procedure was as follows:

The placenta was processed inside a biological safety cabinet. The umbilical cord was first removed, and the amniotic membrane was peeled from the underlying chorionic membrane using blunt dissection. The membrane was rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, N.Y.) to remove gross blood clots and any excess blood cells. The membrane was then washed with 11% anticoagulant citrate dextrose solution (USP) formula A (ACD-A) (Baxter Healthcare Corp., Deerfield, Ill.) in saline (Baxter Healthcare Corp., Deerfield, Ill.) to remove remaining blood cells.

The stromal side of the amnion was cleaned by gently scraping away any remaining connective tissue.

The amnion was then each disinfected in 500 mL of antibiotic solution consisting of gentamicin sulfate (50 μg/mL) (Abraxis Pharmaceutical Products, Schaumburg, Ill.), vancomycin HCI (50 μg/mL) (Hospira Inc., Lake Forest, Ill.), and amphotericin B (2.5 μg/mL) (Sigma Aldrich, St. Louis, Mo.) in DMEM at 37° C.±2° C. for 24-28 hours. Vented caps were used with the incubation flasks. After the incubation period, the membrane was washed with PBS to remove any residual antibiotic solution.

The membrane was mounted on Optitran BA-S 85 reinforced nitrocellulose paper (Whatman, Dassel, Germany) and cut to the appropriate size prior to packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem, N.C.). The stromal side of the amnion was mounted towards the nitrocellulose paper. Once the membrane unit was placed into the FP-90 cryobag and the cryobag was heat sealed, 50 mL of a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. Inverin Co., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, West Lake Village, Calif.) in PlasmaLyte-A (Baxter Healthcare Corp., Deerfield, Ill.) were added through the center tubing line. Any excess air was removed, and the tubing line was subsequently sealed.

The FP-90 cryobag was placed into a mangar bag (10 in.×6 in.) (Mangar Industries, New Britain, PA), which was then heat sealed. The mangar bag was placed into a packaging carton (10.5 in.×6.5 in.×0.6 in.) (Diamond Packaging, Rochester, N.Y.). All cartons were refrigerated at 2-8° C. for 30-60 minutes prior to freezing at −80° C.±5° C. inside a Styrofoam container.

Example 5 Exemplary Manufacturing Process of a Placental Product Comprising Chorionic Membrane and Amniotic Membrane

One method of manufacturing a placental product comprising a chorionic membrane product and an amniotic membrane product according to the presently disclosed manufacturing procedure was as follows:

The placenta was processed inside a biological safety cabinet. The umbilical cord was first removed, and the amniotic membrane was peeled from the underlying chorionic membrane using blunt dissection. Subsequently, the chorion was removed by cutting around the placental skirt on the side opposite of the umbilical cord. The chorion on the umbilical side of the placenta was not removed due to the vascularization on this side. Both membranes were rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, N.Y.) to remove gross blood clots and any excess blood cells. The membranes were then washed with 11% anticoagulant citrate dextrose solution (USP) formula A (ACD-A) (Baxter Healthcare Corp., Deerfield, Ill.) in saline (Baxter Healthcare Corp., Deerfield, Ill.) to remove remaining blood cells.

The chorion was then incubated in 200 mL of a 0.5% dispase (BD Biosciences, Bedford, MA) solution in Dulbecco's modified eagles media (DMEM) (Lonza, Walkersville, Md.) at 37° C.±2° C. for 30-45 minutes to digest the connective tissue layer between the chorion and adjacent trophoblast layer. During this incubation period, the stromal side of the amnion was cleaned by gently scraping away any remaining connective tissue. Once the chorion incubation period was complete, the chorion was rinsed with PBS to remove the dispase solution. Subsequently, the trophoblast layer was removed by gently peeling or scraping away these maternal decidual cells.

The amnion and chorion were then each disinfected in 500 mL of antibiotic solution consisting of gentamicin sulfate (50 μg/mL) (Abraxis Pharmaceutical Products, Schaumburg, Ill.), vancomycin HCl (50 μg/mL) (Hospira Inc., Lake Forest, Ill.), and amphotericin B (2.5 μg/mL) (Sigma Aldrich, St. Louis, Mo.) in DMEM at 37° C.±2° C. for 24-28 hours. Vented caps were used with the incubation flasks. After the incubation period, the membranes were washed with PBS to remove any residual antibiotic solution.

The membranes were mounted on Optitran BA-S 85 reinforced nitrocellulose paper (Whatman, Dassel, Germany) and cut to the appropriate size prior to packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem, N.C.). For the amnion, the stromal side was mounted towards the nitrocellulose paper. Once a membrane unit was placed into the FP-90 cryobag and the cryobag was heat sealed, 50 mL of a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. Inverin Co., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, West Lake Village, Calif.) in PlasmaLyte-A (Baxter Healthcare Corp., Deerfield, Ill.) were added through the center tubing line. Any excess air was removed, and the tubing line was subsequently sealed.

The FP-90 cryobag was placed into a mangar bag (10 in.×6 in.) (Mangar Industries, New Britain, PA), which was then heat sealed. The mangar bag was placed into a packaging carton (10.5 in.×6.5 in.×0.6 in.) (Diamond Packaging, Rochester, N.Y.). All cartons were refrigerated at 2-8° C. for 30-60 minutes prior to freezing at -80° C.±5° C. inside a Styrofoam container.

Example 6 Quantitative Evaluation of Cell Number and Cell Viability after Enzymatic Digestion of Placental Membranes

Amnion and chorion membranes and present placental products (from above) were evaluated for cell number and cell viability throughout the process. These analyses were performed on fresh placental tissue (prior to the antibiotic treatment step), placental tissue post antibiotic treatment, and product units post thaw. Cells were isolated from the placental membranes using enzymatic digestion. For the frozen product units, the FP-90 cryobags were first removed from the packaging cartons and mangar bags. Then the FP-90 cryobags were thawed for 2-3 minutes in a room temperature water bath. Early experiments involved the use of a 37° C.±2° C. water bath. After thaw, the placental membranes were removed from the FP-90 cryobag and placed into a reservoir containing saline (Baxter Healthcare Corp., Deerfield, Ill.) for a minimum of 1 minute and a maximum of 60 minutes. Each membrane was detached from the reinforced nitrocellulose paper prior to digestion.

Amniotic membranes were digested with 40 mL of 0.75% collagenase (Worthington Biochemical Corp., Lakewood, NJ) solution at 37° C.±2° C. for 20-40 minutes on a rocker. After collagenase digestion, the samples were centrifuged at 2000 rpm for 10 minutes. The supernatant was removed, and 40 mL of 0.05% trypsin-EDTA (Lonza, Walkersville, Md) were added and incubated at 37° C.±2° C. for an additional 5-15 minutes on a rocker. The trypsin was warmed to 37° C.±2° C. in a water bath prior to use. After trypsin digestion, the suspension was filtered through a 100 μm cell strainer nylon filter to remove any debris. Centrifugation at 2000 rpm for 10 minutes was performed, and supernatant was removed. Cell pellets were reconstituted with a volume of PlasmaLyte-A that was proportional to the pellet size, and 20 μl of the resuspended cell suspension were mixed with 80 μl of trypan blue (Sigma Aldrich, St. Louis, Mo.) for counting. The cell count sample was placed into a hemocytometer and evaluated using a microscope.

Chorionic membranes were digested with 25 mL of 0.75% collagenase solution at 37° C.±2° C. for 20-40 minutes on a rocker. After collagenase digestion, the suspension was filtered through a 100 μm cell strainer nylon filter to remove any debris. Centrifugation at 2000 rpm for 10 minutes was performed, and supernatant was removed. Cell pellets were reconstituted with a volume of PlasmaLyte-A that was proportional to the pellet size, and 20 μL of the resuspended cell suspension were mixed with 80 μL of trypan blue for counting. The cell count sample was placed into a hemocytometer and evaluated using a microscope.

Placenta membranes were analyzed prior to any processing to determine the initial characteristics of the membranes. Table 1 contains the average cell count per cm² and cell viability values for the amniotic and chorionic membranes from 32 placenta lots.

The average cell count per cm² for the amniotic membrane was 91,381 cells with a corresponding average cell viability of 84.5%. For the chorionic membrane, the average cell count per cm² was 51,614 cells with a corresponding cell viability of 86.0%.

These data illustrate cell numbers that are useful with certain embodiments of the present invention; e.g. placental product comprising an amniotic membrane containing about 10,000 to about 360,000 cells/cm². Since the amniotic membrane consists of epithelial cells and stromal cells, experiments were conducted to determine the ratio of epithelial cells to stromal cells. Amniotic membranes from 3 placenta lots were analyzed. First, a 5 cm×5 cm piece of amniotic membrane was digested with approximately 25 mL of 0.05% trypsin-EDTA (Lonza, Walkersville, Md.) at 37° C.±2° C. in a water bath for 30 minutes. After the incubation step, epithelial cells were removed by gently scraping the cells from the membrane. After rinsing with PBS (Gibco Invitrogen, Grand Island, N.Y.), the membrane was subsequently digested in the same manner as chorionic membrane (described above). In addition, another intact 5 cm×5 cm piece of amniotic membrane was digested using the standard procedure (described above) to determine the total number of cells. The percentage of stromal cells was then determined by dividing the cell count from the amniotic membrane with the epithelial cells removed with the cell count from the intact membrane.

Results indicate that 19% of the total cells were stromal cells. Therefore, approximately 17,362 stromal cells were present in amniotic membrane with approximately 74,019 epithelial cells. These data indicated that there are approximately 3 times more stromal cells in chorionic membranes as compared to amniotic membranes. This ratio is consistent with certain embodiments of the present invention that provide a placental product comprising a chorionic membrane and an amniotic membrane, wherein the chorionic membrane comprises about 2 to about 4 times more stromal cells relative to the amniotic membrane. Table 1 Cell count per cm² and cell viability values from fresh placental tissue from 32 donors.

Cell Membrane Statistics Count per cm² Cell Viability Amnion Average 91,381 84.5% SD 49,597 3.7% Chorion Average 51,614 86.0% SD 25,478 4.7%

Cell count and cell viability was assessed after the antibiotic treatment step. Table 2 provides the results from these analyses. Cell recoveries from this step for the amniotic membrane and the chorionic membrane were 87.7% and 70.3%, respectively.

TABLE 2 Cell count per cm², cell viability, and process (antibiotic treatm^(e)nt) cell recovery values for post antibiotic placental tissue from 28 donors. Cell Count per Process Cell Membrane Statistics cm² Cell Viability Recovery Amnion Average 75,230 84.4% 87.7% SD 46,890 4.2% 49.4% Chorion Average 33,028 85.6% 70.3% SD 18,595 4.4% 31.1%

Example 7 Development of a Placental Product Cryopreservation Procedure

Cryopreservation is a method that provides a source of tissues and living cells. A main objective of cryopreservation is to minimize damage to biological materials during low temperature freezing and storage. Although general cryopreservation rules are applicable to all cells, tissues, and organs, optimization of the cryopreservation procedure is required for each type of biological material. The present application discloses a cryopreservation procedure for placental membrane products that can selectively deplete immunogenic cells from the placental membranes; and preserve viability of other beneficial cells that are the primary source of factors for the promotion of healing.

During cryopreservation method development for placental membranes, the present inventors evaluated key parameters of cryopreservation including volume of cryopreservative solution, effect of tissue equilibration prior to freezing, and cooling rates for a freezing procedures.

Acceptance of tissue allografts in the absence of immunosuppression will depend on the number of satellite immune cells present in the tissue. Cryopreservation is an approach which can be utilized to reduce tissue immunogenicity. This approach is based on differential susceptibility of different cell types to freezing injury in the presence of DMSO; leukocytes are sensitive to fast cooling rates. The freezing rate of 1° C./min is considered optimal for cells and tissues including immune cells. Rapid freezing rates such as 60-100° C./min eliminate immune cells. However, this type of procedure is harmful to other tissue cells, which are desirable for preservation according to the present invention. The developed cryopreservation procedure utilized a cryopreservation medium containing 10% DMSO, which is a key component protecting cells from destruction when water forms crystals at low temperatures. The second step of cryopreservation was full equilibration of placental membrane in the cryopreservation medium, which was achieved by soaking membranes in the cryopreservation medium for 30-60 min at 4° C. This step allowed DMSO to penetrate the placental tissues. Although there are data in the literature showing that tissue equilibration prior to freezing affects survival of immune cells (Taylor & Bank, Cryobiology, 1988, 25:1),

It was unexpectedly found that 30-60 min placental membrane equilibration in a DMSO-containing solution at 2-8° C. increases sensitivity of immune cells to freezing so that these type of cells cannot withstand freezing even at a 1° C./min freezing rate.

Temperature mapping experiments were performed to analyze the temperature profiles of potential cryopreservation conditions for the membrane products. These results are illustrated in FIG. 1. Eight (8) FP-90 cryobags were filled with either 20 mL or 50 mL of cryopreservation solution, and temperature probes were placed inside each cryobag. The first set of parameters (conditions 1 through 4 of FIG. 1 a through FIG. 1 d, respectively) involved a 30-minute refrigeration (2-8° C.) step prior to freezing (−80° C.±5° C.). In addition, the analysis involved freezing of the cryobags either inside a Styrofoam container or on the freezer shelf. The second set of parameters (conditions 5 through 8 of FIG. 1 e through FIG. 1 h, respectively) involved direct freezing (−80° C.±5° C.) of the cryobags either inside a Styrofoam container or on the freezer shelf. The results indicated that condition 6 and condition 2 exhibited the most gradual temperature decreases. Gradual temperature decreases are typically desired in order to preserve cell viability. The difference between condition 6 and condition 2 was that condition 2 included a 30-minute refrigeration step. Therefore, the decrease in temperature from the start of freezing to -4° C., where latent heat evolution upon freezing occurs, was examined further. For condition 6, the rate of cooling was approximately—1° C./minute during this period. The rate of cooling for condition 2 was approximately—0.4° C./minute during the same timeframe. Therefore, condition 2 was selected for incorporation into a non-limiting cryopreservation process since slower rates of cooling are generally desired to maintain optimal cell viability.

FIG. 2 depicts the effects of cryopreservation solution volume on process (cryopreservation) cell recovery for the amniotic membrane. The analysis of the 10 mL cryopreservation solution volume involved 5 placenta lots, and the analysis of the 20 mL cryopreservation solution volume included 3 lots. For the 50 mL cryopreservation solution volume, 14 placenta lots were analyzed.

As depicted in FIG. 2, the 50mL volume of cryopreservation solution volume provided superior cell recovery compared to that of the 10 ml and 20 ml. These data indicate that a cryopreservation medium volume of greater than 20mL such as about 50 mL or more can provide superior placental product according to the present invention.

FIG. 3 shows the results of a similar study of analysis of the cryopreservation cell recovery for the chorionic membrane, demonstrating that a cryopreservation solution volume of 50 mL was optimal. The analysis of the 10 mL cryopreservation solution volume involved 5 placenta lots, and the analysis of the 20 mL cryopreservation solution volume included 3 lots. For the 50 mL cryopreservation solution volume, 16 placenta lots were analyzed.

Experiments were conducted to evaluate different potential freezing conditions to maximize cell recovery after the cryopreservation process. FIG. 4 (cells from amniotic membrane) and FIG. 5 (cells from chorionic membranes) depict these results, showing the effects of refrigeration time and freezing parameters on process (cryopreservation) cell recovery for the chorionic membrane. Three conditions were analyzed. These conditions were also linked to the temperature mapping studies. The first condition involved directly freezing the product unit on a shelf within the freezer (−80° C.±5° C.). The second condition also contained a direct freeze, but the product unit was placed into a Styrofoam container within the freezer. The third condition included a refrigeration (2-8° C.) period of 30 minutes prior to the freezing step. For the amniotic membrane, 3 placenta lots were evaluated. Two (2) placenta lots were analyzed for the chorionic membrane. Results indicated that the third condition was optimal for both membrane types.

All of the cryopreservation parameters that were assessed for the amniotic and chorionic membranes are summarized in Table 3 and Table 4. The evaluation of the cell recoveries and cell viabilities from these experiments resulted in the selection of the final parameters for the manufacturing process. In addition, all average cell viability values were 70%.

TABLE 3 Post thaw cell count per cm², cell viability, and process (cryopreservation) cell recovery values for the amniotic membrane. Cell Process Condition Count Cell Cell Comments/ Parameter Tested Statistics per cm² Viability Recovery Conclusions Refrigerate All Average 55,709 83.4% 64.2% Overall at 2-8° C. for conditions SD 45,210 4.4% 22.5% assessment. 30-60 min N 32 32 32 and freeze at −80° C. ± 10° C. Refrigeration 30 min Average 52,173 83.1% 63.7% No significant time interval SD 39,750 4.5% 21.4% difference N 26 26 26 found in 60 min Average 71,033 85.0% 66.5% process cell SD 66,525 3.9% 29.3% recovery. A N 6 6 6 30-60 min range was established. Thawing 37° C. ± Average 48,524 83.3% 64.0% No significant temperature 2° C. water SD 27,804 1.7% 34.4% difference bath N 7 7 7 found in Room Average 57,721 83.5% 64.3% process cell temp SD 49,271 4.9% 19.0% recovery. The water bath N 25 25 25 room temp condition was selected for logistical reasons. Holding 1-15 min Average 50,873 83.1% 65.0% No significant period after SD 38,969 3.9% 24.2% difference transfer into N 26 26 26 found in saline 1 hr Average 76,667 85.1% 61.0% process cell SD 66,565 6.2% 14.3% recovery. N 6 6 6 Membranes can be held in saline for up to 1 hr. Tissue size 5 cm × 5 Average 58,431 83.3% 62.8% No decrease cm SD 47,603 4.5% 21.7% in process cell N 28 28 28 recovery from 2 cm × 2 Average 36,656 84.4% 73.9% the 5 cm × 5 cm SD 13,175 3.4% 29.5% cm product to N 4 4 4 the 2 cm × 2 cm product. Both sizes were acceptable for use.

TABLE 4 Post thaw cell count per cm², cell viability, and process (cryopreservation) cell recovery values for the chorionic membrane. Cell Process Condition Count Cell Cell Parameter Tested Statistics per cm² Viability Recovery Refrigerate All Average 23,217 87.3% 102.8% at 2-8° C. for conditions SD 9,155 4.1% 65.5% 30-60 min N 27 27 27 and freeze at −80° C. ± 10° C. Dispase 30 min Average 22,354 85.7% 81.1% No decrease treatment SD 9,505 5.1% 32.4% in process cell N 24 24 24 recovery for 45 min Average 27,125 90.6% 172.6% the 45 min SD 7,963 2.2% 101.2% treatment. A N 6 6 6 30-45 min range was established. Refrigeration 30 min Average 23,815 86.8% 102.2% The process time interval SD 9,681 5.2% 68.8% recovery value N 25 25 25 was >80% for 60 min Average 20,773 85.8% 84.9% the 60 min SD 7,356 4.7% 14.4% time interval. N 5 5 5 A 30-60 min range was established. Thawing 37° C. ± Average 33,360 85.9% 114.7% No significant temperature 2° C. water SD 8,497 4.0% 38.1% difference bath N 5 5 5 found in Room Average 21,298 86.8% 96.3% process cell temp SD 8,189 5.3% 67.2% recovery. The water bath N 25 25 25 room temp condition was selected for logistical reasons. Holding 1-15 min Average 23,733 86.6% 100.6% No significant period after SD 9,674 5.1% 67.0% difference transfer into N 26 26 26 found in saline 1 hr Average 20,550 87.0% 91.4% process cell SD 6,575 4.8% 32.0% recovery. N 4 4 4 Membranes can be held in saline for up to 1 hr. Tissue size 5 cm × 5 Average 23,391 86.1% 99.6% No decrease cm SD 8,865 5.0% 58.7% in process cell N 23 23 23 recovery from 2 cm × 2 Average 23,036 88.4% 98.7% the 5 cm × 5 cm SD 11,362 5.0% 81.3% cm product to N 7 7 7 the 2 cm × 2 cm product. Both sizes were acceptable for use. Notes: cm = centimeter; min = minutes; temp = temperature; hr = hour, SD = standard deviation; N = number.

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing about 40,000 to about90,000 or to about 260,000 cells/cm².

Example 8 Qualitative Evaluation of Cell Viability by Tissue Staining

The amniotic and chorionic membranes were stained using a LIVE/DEAD® Viability/Cytotoxicity kit (Molecular Probes Inc., Eugene, Oreg.) to qualitatively assess cell viability. Staining was performed as per the manufacturer's protocol. Membrane segments of approximately 0.5 cm×0.5 cm were used. Evaluation of stained membranes was performed using a fluorescent microscope. An intense uniform green fluorescence indicated the presence of live cells, and a bright red fluorescence indicated the presence of dead cells. Images of fresh amniotic and chorionic membranes as well as cryopreserved amniotic and chorionic membranes demonstrated that the manufacturing process did not alter the phenotypic characteristics of the membranes post thaw.

FIG. 6 contains representative images of fresh amniotic and chorionic membranes as well as cryopreserved amniotic and chorionic membranes. These images demonstrated that the manufacturing process did not alter the phenotypic characteristics of the membranes and the proportion of viable cell types (epithelial and stromal cells) in the membranes post thaw.

FIG. 6. Representative images of the live/dead staining of the epithelial layer of fresh amniotic membrane (A); epithelial layer of cryopreserved amniotic membrane (B); stromal layer of fresh amniotic membrane (C); stromal layer of cryopreserved amniotic membrane (D); fresh chorionic membrane (E); and cryopreserved chorionic membrane (F). Live cells are green, and dead cells are red.

Example 9 Placental Tissue Immunogenicity Testing

One unique feature of the human amnion and chorion is the absence of fetal blood vessels that prevent mobilization of leukocytes from fetal circulation. On the fetal side, macrophages resident in the chorioamniotic mesodermal layer represent the only population of immune cells. Thus, fetal macrophages present in the chorion and amnion are the major source of tissue immunogenicity. However, the number of macrophages in amnion is significantly lower (Magatti et al, Stem Cells, 2008, 26: 182), and this explains the low immunogenicity of amnion and the ability to use it across HLA barriers without matching between the donor and recipient (Akle et al, Lancet, 1981, 8254:1003; Ucakhan et al., Cornea, 2002, 21:169). In contrast, the chorion is considered immunogenic. In a study where the amnion was used together with the chorion for plastic repair of conjunctival defects, the success rate was low (De Roth Arch Ophthalmol, 1940, 23: 522). Without being bound by theory, the present inventors believe that removal of CD14+ cells from placental membranes eliminates activation of lymphocytes in vitro. In addition to the presence of fetal macrophages, the present inventors believe that immunogenicity of chorion can be mediated by contamination of blood cells coming from the maternal trophoblast, which contains blood vessels. Thus, the processing of placental membrane for clinical use can be enhanced by purification of the amnion and chorion from maternal trophoblasts and selective elimination of all CD14+ fetal macrophages.

Immunogenicity testing can be used to characterize a placental product as safe clinical therapeutics. For example, two bioassays can be used to test immunogenicity of manufactured placental products: Mixed Lymphocyte Reaction (MLR) and Lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-α secretion.

Example 10 Mixed Lymphocyte Reaction (MLR)

An MLR is a widely used in vitro assay to test cell and tissue immunogenicity. The assay is based on the ability of immune cells (responders) derived from one individual to recognize allogeneic Human Leukocyte Antigen (HLA) and other antigenic molecules expressed on the surface of allogeneic cells and tissues (stimulators) derived from another individual when mixed together in a well of an experimental tissue culture plate. The response of immune cells to stimulation by allogeneic cells and tissues can be measured using a variety of methods such as secretion of particular cytokines (e.g., Interleukin (IL)-2), expression of certain receptors (e.g., IL-2R), or cell proliferation, all of which are characteristics of activated immune cells.

Placental tissue samples representing different steps of the presently disclosed manufacturing process were used for immunogenicity testing. These samples included amnion with chorion and trophoblast as a starting material and separated choriotrophoblast, chorion, trophoblast, and amnion. Both freshly purified and cryopreserved (final products) tissues were tested.

For the MLR assay, cells from placental tissues were isolated using 280 U/mL of collagenase type II (Worthington, Cat No. 4202). Tissues were treated with enzyme for 60-90 min at 37° C.±2° C., and the resulting cell suspension was filtered through a 100 μm filter to remove tissue debris. Single cell suspensions were then centrifuged using a Beckman, TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant was discarded after each wash, and cells were resuspended in 2 mL of DMEM (Invitrogen, Cat No. 11885) and evaluated for cell number and cell viability by counting cells in the presence of Trypan blue dye (Invitrogen, Cat No. 15250-061). For the MLR, placental-derived cells were mixed with allogeneic hPBMCs at a 1:5 ratio in 24-well culture plates in DMEM supplemented with 5% fetal bovine serum (FBS) and incubated for 4 days in the incubator containing 5% CO₂, 95% humidity at 37° C.±2° C. Human Peripheral Blood Mononuclear Cells (hPBMCs) alone were used as a negative control, and a mixture of two sets of hPBMCs derived from two different donors was used as a positive MLR control. After 4 days of incubation, cells were collected from wells, lysed using a lysis buffer (Sigma, Cat No. C2978) supplemented with protease inhibitor cocktail (Roche, Cat No. 11836153001), and IL-2R was measured in cell lysates using the sIL-2R ELISA kit (R&D Systems, Cat No. SR2A00) according to the established protocol (G-SOP-Q088).

The level of IL-2R is a measure of activation of T-cells in response to immunogenic molecules expressed by allogeneic cells. Results of 2 out of 12 representative experiments are shown in FIG. 7 and FIG. 8. Results presented in these figures demonstrate a method of manufacture of placental membranes, resulting in low immunogenicity of the final products.

As depicted in FIG. 7: the manufacturing process serially reduces immunogenicity of the placental product. Samples representing different steps of the manufacturing process (Chorion+Trophoblast (CT), Trophoblast (T), Amnion (AM), and Chorion (CM)) were co-cultured with hPBMCs for 4 days. IL-2αR was measured in cell lysates as a marker of T-cell activation. Negative control shows a basal level of immune cell activation: PBMCs derived from one donor were cultured alone. Positive control: a mixture of PBMCs derived from 2 different donors.

As depicted in FIG. 8, selective depletion of immunogenicity results from the present cryopreservation process of producing the present placental products, as evidenced by the significant decrease in immunogenicity upon cryopreservation.

Example 11 LPS-Induced TNF-α Secretion by Placental Membrane Cells

As described herein, fetal macrophages present in the amnion and chorion are a major source of tissue immunogenicity. Without being bound by theory, the present inventors believe that removal of CD14+ cells from placental membrane eliminates activation of lymphocytes and that depletion of allogeneic donor tissue macrophages decreases the level of inflammatory cytokine secretion and tissue immunogenicity. The inventors also believe that reduction of tissue immunogenicity can also be reached by depletion of TNF-α with anti-TNF-α antibodies or suppression of TNF-α secretion by IL-10. Macrophages in fetal placental membranes respond to bacteria by secretion of inflammatory cytokines. The secretion of TNF-α by fresh placental membranes in vitro in response to bacterial LPS is significantly higher in the chorionic membrane. Thus, the present inventors believe that immunogenicity of placental membranes is mediated by macrophages, the amount and/or activity of which is higher in the chorionic membrane.

According to the present invention, selective depletion of macrophages is an approach to selectively deplete immunogenicity of the amniotic and chorionic membranes, allowing the use of both allogeneic membranes for clinical applications. The assay of functional macrophages in a placental product is used here as an assay for immunogenicity testing (e.g. in production or prior to clinical use) based on the facts that: macrophages are the source of immunogenicity in chorionic membranes. Macrophages in placenta-derived membranes respond to bacterial LPS by secretion of high levels of TNF-α; and TNF-α0 is a critical cytokine involved in immune response and allograft tissue rejection. Therefore, secretion of TNF-α by placenta-derived membranes in response to LPS is used here to characterize tissue immunogenicity and for pre-use screening.

Example 12 Establishment of Allowed LPS-Induced TNF-α Secretion Level by Placental Membranes

Data from published reports regarding the level of TNF-α, which is associated with the absence or an insignificant immune response in a variety of experimental systems, are presented in Table 5. These data indicate that a TNF-α level below 100 pg/mL correlates with a low immune response. The ability of amniotic and chorionic membranes to produce TNF-α spontaneously and in response to bacteria or bacterial LPS in vitro has been shown by a number of investigators. Table 6 summarizes such data. The lowest spontaneous TNF-α secretion by amniotic membrane of about 70 pg/cm² of the membrane was reported by Fortunato et al. All reports also showed that fresh placental membranes secrete large amounts of TNF-α in response to bacteria or bacterial LPS, which is attributed to the presence of viable functional macrophages.

TABLE 5 TNF-α levels associated with no or an insignificant immune response. TNF-α levels associated with the Description of absence/reduction experimental system of immune response Comments References IL-10-induced inhibition Mean 260 pg/mL Wang et al., of MLR in vitro. Transplantation, TNF was measured in 2002, 74: 772 tissue culture supernatant by ELISA. MLR using skin tissue Mean 100 pg/mL explants (0.02 cm2 per well) as stimulators in the presence or absence of IL-10 (skin explant assay). Skin tissue destruction was assessed microscopically, and severity was assigned based on histopathological tissue damage. Endogeneous TNF ~0.04 U/mL for the TNF activity Shalaby et al, J production in MLR in the negative control and per mg is not Immunol, 1988, presence or absence of MLR in the presence provided. 141: 499 anti-TNF antibodies. TNF of anti-TNF levels were assessed antibodies, which using the WEHI-164 correlated with no or cytotoxicity assay. significant inhibition of lymphocyte proliferation TNF levels in BAL fluid of Isograft: below Unmodified Sekine et al, J lung isografts, unmodified detection; allograft: ~45 pg/mL Immunol, 1997, allograft, and alveolar AM-depleted allograft: (immunogenic) 159: 4084. macrophages (AM) ~15 pg/mL of BAL depleted allograft in rats. (total 75 pg/5 ml of BAL) TNF levels in MLR after ~<200 pg/mL TNF Ohashi et al, 48 hours in the presence correlated with a Clin Immunol, or absence of advanced complete inhibition of 2009, e-pub glycation end products MLR ahead of print (MLR inhibitors). TNF levels in MLR. <100 pg/mL TNF in Toungouz et al, MLR with HLA- Hum Immunol, matched donors 1993, 38: 221 (control, no stimulation) TNF activity in MLR when Negative control ~20 Unit of activity Lomas et al, pieces of cryopreserved U of TNF activity; was calculated Cell Tissue skin allografts (~0.2 cm²) MLR with skin as TNF in Bank, 2004, 5: were incubated with explants - 0-40 U; ng/mL divided 23. hPBMCs for 24 hours. Positive control - 600 U by OD at 570 nm Positive control: for the hPBMC + LPS; negative - same hPBMC alone. experimental well Cytokine time course in Optimal TNF Jordan & Ritter, MLR, including TNF. after 24 hours - J Immunol ~150 pg/mL Meth, 2002, 260: 1 MLR using skin tissue For no skin Recalculation Dickinson et al, explants (0.02 cm² per destruction: 0.5-1.1 pg/mL per 1 cm² of Cytokine, 1994, well) as stimulators in the for HLA skin tissue: 6: 141 presence or absence of compatible lowest TNF anti-TNF antibodies (skin responders, and 2.6-1376 pg/mL non- explant assay). Skin for immunogenic tissue destruction was unmatched MLR level is 100 pg/cm² assessed microscopically, and severity was assigned based on histopathological tissue damage.

TABLE 6 Secretion of TNF in vitro by fresh amniotic and chorionic membranes. TNF levels Comments/ secreted by fresh recalculations placental of the lowest Description of membranes in TNF levels per experimental system culture cm² References TNF secretion by Chorion: basal Lowest TNF Zaga et al., Biol “fresh” amnion and 3.3 ± 0.46 ng/cm², level for amnion Reprod, 2004, chorion tissues (1.44 cm2) LPS-induced - 150-250 ng/cm² is 1200 pg/cm² 71: 1296 incubated for 24 Amnion: basal hours in the presence 2.5 ± 1.3 ng/cm², or absence of LPS LPS-induced - ~50 ng/cm² (500 ng/mL). TNF secretion by Basal ~1-2.5 pg/μg Lowest TNF Zaga-Clavellina “fresh” amnion and total protein in the level for amnion et al, Reprod chorion tissues (1.8 cm medium for both is 800 pg/cm² Biol Endocrinol, diameter disks: 2.5 cm²) amnion and 2007, 5: 46 incubated for 24 chorion; hours in the presence E. Coli-induced: or absence of E. Coli amnion - 29.2 in 1 mL medium. (14.5-35.3) pg and chorion - 53.15 (40-94.2) pg per μg total protein TNF secretion by Basal: ~2-64 U/mL 1 unit = ~100-200 pg/mL; Paradowska et “fresh” amnion and or 8-10 mg chorion; Lowest TNF al, Placenta, chorion tissues <1 U/mL for 5-7 mg level for amnion 1997, 18: 441 (chorion 8-10 mg amnion. is <100 pg/mL tissue/mL; amnion 5-7 mg/mL, LPS-induced: >100 corresponding to 0.02-0.04 cm²) U/10 mg for chorion <2500 pg/cm² incubated for 20 hours and ~15-17 U/10 mg in the presence or for amnion absence of LPS (5 μg/mL). TNF secretion by Amnion: Basal - 40 pg/mL, Lowest TNF Fortunato et al, “fresh” amnion (0.57 cm²) LPS-induced - 410 pg/mL level for fresh Am J Obstet in 0.8 mL amnion is ~70 pg/cm² Gynecol, 1996, incubated for 24 hours 174: 1855 in the presence or absence of LPS (50 ng/mL). TNF secretion by Basal: Amnion ~7-13 ng/mL/g Amnion is 5-7 mg Thiex et al, “fresh” amnion and tissue); corresponds Reprod Biol chorion tissues (4 cm²) Chorion ~18 ng/mL/g ~0.02-0.04 cm²; Endocrinol, incubated for 24 tissue 1 g is ~6 cm²; 2009, 7: 117 hours in the presence LPS-induced (1000 ng/mL): Lowest TNF or absence of LPS (1-1000 ng/mL) Amnion level for amnion ~14 ng/mL/g), is ~1000 pg/cm² Chorion ~27 ng/mL/g

Example 13 LPS-Induced TNF-α Secretion and CT induced MLR Immunogenicity Assay

2 cm×2 cm pieces of placental membranes representing intermediates and final products were placed in tissue culture medium and exposed to bacterial LPS (1 μg/mL) for 20-24 hr. After 24 hours, tissue culture supernatant were collected and tested for the presence of TNF-α using a TNF-α ELISA kit (R&D Systems) according to the manufacturer's protocol. Human hPBMCs (SeraCare) known to contain monocytes responding to LPS by secretion of high levels of TNF-α were used as a positive control in the assay. hPBMCs and placental tissues without LPS were also included as controls in the analysis. In this assay, TNF detected in the culture medium from greater than 70 μg/cm² (corresponding to 280 pg/mL) for both spontaneous and LPS-induced TNF-α secretion was considered immunogenic.

As depicted in FIG. 9A and FIG. 9B, the manufacturing process serially reduces immunogenicity of the placental product. Samples representing different steps of the manufacturing process (Amnion+Chorion+Trophoblast (ACT), Chorion+Trophoblast (CT), Amnion (AM), and Chorion (CM)) were incubated in the presence of LPS for 24 hr, and after that tissue culture supernatants were tested for the TNF-α by ELISA. Tissues cultured in medium without LPS show the basal level of TNF a secretion. PBMCs, which are known to secrete high levels of TNF, were used as a positive control.

The low levels of TNF-α and the absence of the response to LPS by AM and CM indicates the absence of viable functional macrophages that are the major source of immunogenicity for amniotic and chorionic membranes. Results of this assay showed a correlation with the MLR data: tissues that produce high levels of TNF-α in response to LPS are immunogenic in the MLR assay (FIG. 9A and FIG. 9B, for TNF-α secretion; FIG. 9, C-MLR).

As depicted in FIG. 9A and FIG. 9B, the manufacturing process serially reduces immunogenicity of the placental product. Samples representing different steps of the manufacturing process (Amnion+Chorion+Trophoblast (ACT), Chorion+Trophoblast (CT), Amnion (AM), and Chorion (CM)) were incubated in the presence of LPS for 24 hr, and after that tissue culture supernatants were tested for the TNF-α by ELISA. Tissues cultured in medium without LPS show the basal level of TNF a secretion. PBMCs, which are known to secrete high levels of TNF, were used as a positive control.

Choriotrophoblast (CT), which secreted high levels of TNF-α (FIG. 9, B), was tested in MLR against two different PBMC donors. CT cells were co-cultured with PBMCs for 4 days. IL-2αR was measured in cell lysates as a marker of T-cell activation. Positive control: a mixture of PBMCs derived from 2 different donors.

FIG. 9C shows that preparations producing high levels of TNF-α are immunogenic. Choriotrophoblast (CT), which secreted high levels of TNF-α (FIG. 9 B), was tested in MLR against two different PBMC donors. CT cells were co-cultured with PBMCs for 4 days. IL-2αR was measured in cell lysates as a marker of T-cell activation. Positive control: a mixture of PBMCs derived from 2 different donors.

Example 14 Analysis of Placental Cells by FACS

Knowing the cellular composition of amnion and chorionic membranes is important for developing a thorough understanding of potential functional roles in wound healing and immunogenicity. Previous reports demonstrated that both amnion and chorion contains multiple cell types. Purified amnion has two major cellular layers: epithelial cells and stromal. In addition to epithelial cells and fibroblasts, stromal cells were identified in the amnion and chorion. Although there are no fetal blood vessels within either the amniotic or chorionic membranes, both membranes comprise resident fetal macrophages. The close proximity to maternal blood circulation and decidua provide a potential source of immunogenic cells (maternal leukocytes and trophoblast cells) and therefore are a potential source of immunogenicity. To investigate the cellular composition of the amnion and chorion, FACS analysis was performed.

Example 14.1 FACS Procedure: Single Cell Suspension Preparation

Purified amnion and chorionic membranes were used for cellular phenotypic analysis via FACS. Cells from amnion and chorion were isolated using 280 U/mL collagenase type II (Worthington, Cat No. 4202). Tissues were treated with enzyme for 60-90 min at 37° C.±2° C., and the resulting cell suspension was filtered through a 100 μm filter to remove tissue debris. Single cell suspensions were then centrifuged using a Beckman TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant was discarded after each wash, and cells were resuspended in 2 mL of FACS staining buffer (DPBS+0.09% NaN₃+1% FBS).

Example 14.2 Immunolabeling Cells for Specific Cellular Markers

Once the single cell suspension was prepared according to Example 10, a minimum of 1×10⁵ cells in 100 μL of FACS staining buffer was treated with antibodies labeled with fluorescent dye. Table 7 provides descriptions of the antibodies and the amounts used. For cell surface markers, cells were incubated for 30 min at room temperature in the dark with antibodies followed by washing twice with FACS staining buffer by centrifugation at 1300 rpm for 5 min using a Beckman TJ-6 centrifuge. Cells were then resuspended in 400 μL of FACS staining buffer and analyzed using a BD FACSCalibur flow cytometer. To assess cell viability, 10 μL of 7-AAD regent (BD, Cat No. 559925) was added just after the initial FACS analysis and analyzed again. For intracellular staining, cells were permeabilized and labeled following the manufacturer's recommendations (BD Cytofix/Cytoperm, Cat No. 554714) and analyzed using a BD FACSCalibur flow cytometer.

TABLE 7 Description of reagents used for placental cell characterization by FACS. Volume of Cell marker antibody antibody and solution Cell marker Cell marker label type Cat No. used type specificity IgG1 BD 559320  5 μL Cell surface Isotype control isotype- PE CD105-PE Caltag 20 μL Cell surface MSC marker MHCD10504 CD166-PE BD 559263 80 μL Cell surface MSC marker CD45-PE BD 555483 10 μL Cell surface Hematopoetic cell marker IgG2a BD 555574  2 μL Cell surface Isotype control isotype- PE CD14-PE BD 555398 20 μL Cell surface Monocyte marker HLA-DR-PE BD 556644 20 μL Cell surface HLA class II specific for antigen- presenting cells IgG1 BD555748  5 μL Cell surface Isotype control isotype- FITC CD86-FITC BD 557343 20 μL Cell surface Immune co- stimulatory marker CD40-FITC BD 556624 20 μL Cell surface Immune co- stimulatory marker IgG1 Dako X0931 10 μL Intracellular Isotype control isotype- unlabeled Cytokeratin Dako M7018  2 μL Intracellular Trophoblast 7-unlabeled marker Rabbit anti- Dako F0261  5 μL Intracellular Secondary mouse FITC antibody

Example 15 Phenotypic Analysis of Placental Cells

FACS analysis of single cell suspensions of both amnion and chorion membranes demonstrates that both membranes contain cells expressing markers specific for mesenchymal stem cells (refer to Table 8), implicating the presence of MSCs. In addition, several immunogenic markers, which are more likely expressed on CD14+ placental macrophages, were detected. The % ranges for different markers are wide. It can be explained by: 1) high variability in cell number between placenta donors; and 2) technical issues, which include the presence of high and variable cellular and tissue debris in the cellular suspension. Although debris can be gated out, debris particles that are comparable with cells by size will affect the accuracy of the calculated % for each tested marker. In addition, Table 9 provides a FACS analysis of cells isolated from the amniotic and chorionic membranes that were cultured in 10% FBS in DMEM at 37° C.±2° C. until confluency (passage 0 cells).

These data demonstrated that cells derived from amniotic and chorionic membranes retained a phenotype similar to MSCs after culturing. In conclusion, the presence of stromal cells in placental tissues was confirmed by FACS analysis.

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing MSCs.

TABLE 8 Characterization of the cellular composition of placental membranes based on selective CD markers. Amnion (% Chorion (% Marker range) range) MSC Markers CD105 72.1-88.2  6.4-78.5 CD166 17.3-58.0  4.8-51.5 Hematopoietic Cell CD14 6.93-10.5 0.9-6.1 Markers CD45 4.4-9.9  4.6-14.7 Immune co-stimulatory HLA-DR   0-5.6   0-14.7 markers CD86 24.3-49.6  4.9-22.5 CD40  7.0-68.7   2-5.8 Trophoblast marker Cytokeratin-7 1.36-4.66  2.71-23.07

TABLE 9 FACS analysis of cultured cells (passage 0) from placenta lot D16. Cell Surface Marker Amnion (%) Chorion (%) CD45 2.18 0.53 CD166 92.77 82.62 CD105 83.02 86.73 CD49a 92.28 92.26 CD73 89.57 94.57 CD41a −0.03 −0.05 CD34 −0.23 −0.25 HLA-DR −0.23 −0.19 CD19 −0.19 −0.22 CD14 −0.25 −0.27 CD90 99.12 98.00

Example 16 Aherence of Cells Derived from Placental Products.

Therapeutic cells, in optional embodiment of the present invention, are adherent, express specific cellular markers such as CD105 and lack expression of other markers such as CD45, and demonstrate the ability to differentiate into adipocytes, osteoblasts, and

The expression of specific cellular markers has already been described in Example 15. To determine if the cells within the placental product derived from the chorionic membrane can adhere to plastic and differentiate into one of the lineages, cells were isolated from the placental product derived from the amnion as described in this invention and cultured at 37° C.±2° C. and expanded.

FIG. 10 shows representative images of cells isolated and cultured from amniotic (FIG. 10.A) and chorionic (FIG. 10.B) membranes demonstrating plastic adherence, which is a key feature of MSCs. As a comparison, a representative image of MSCs isolated and expanded from human bone marrow aspirate is also provided (FIG. 10.C). Together, these data show that cells derived from amniotic and chorionic membranes retain a phenotype similar to MSCs after culturing as demonstrated by the cellular markers present in addition to the ability of the cells to adhere to plastic. In conclusion, the presence of MSCs in placental tissues was confirmed by FACS analysis and tissue culture

Example 17 Live CD45+ FACS Analysis

As CD45 is a general marker for hematopoietic cells and therefore a marker for the presence immunogenic cells, the presence of CD45+ cells may correlate well with how immunogenic a tissue may be. An initial study indeed showed a correlation between amount of immunogenicity as measured via an in vitro MLR assay of placental tissue at various stages within the manufacturing process (as described previously), and the amount of CD45+ cells was determined via FACS analysis. As FIG. 11 demonstrates, membranes that trigger the expression of high levels of IL-2sR on hPBMC responders in MLR also contained a high percentage of CD45+ cells, indicating that immunogenicity of placental membranes can be correlated with the number of CD45+ cells. Further studies revealed, however, that quantifying CD45+ cells via FACS alone showed high variability that did not allow for the establishment of a safety threshold for CD45+ cells in placental membranes. Accordingly, the inventors evaluated whether or not viability of CD45+ cells is correlated with immunogenicity.

To eliminate some of the variability in CD45+ measurements via FACS, viability of CD45+ cells was assessed, as dead CD45+ cells do not contribute to immunogenicity. To ensure an accurate assessment of live CD45+ cells, a pilot experiment was conducted in which a single cell suspension of amnion membrane was spiked in with a known concentration of live CD45+ cells (hPBMCs) ranging from a theoretical 1.25% to 20% (0.75-12%—actual % of the spiked cells) of the total cell concentration in suspension. Cells were stained with CD45-PE antibody at determined concentrations (refer to Table 10), incubated with 7-AAD cell viability test reagent, and analyzed using a BD FACSCalibur. Table 10 demonstrates that recovery of known amounts of CD45+ cells was not correct (4th column in the table). For example, although 12% of PBMCs was spiked into a single-cell suspension of amnion membrane, only 4.26% of CD45+ cells were recovered according to FACS analysis (>60% difference from the actual spike). To correlate with immunogenicity, MLR was also performed in parallel. Briefly, single cell suspensions of amniotic membrane spiked with various amounts of live hPBMCs were co-cultured with another donor of PBMCs in the MLR. FIG. 12 depicts a correlation between the amount of CD45+ cells present in amnion-derived cell suspensions and immunogenicity in MLR in vitro. Table 10 and FIG. 12 show that the suspensions spiked with higher amounts of live CD45+ cells resulted in higher immunogenicity as measured by IL-2sR expression on the hPBMC responder donor.

TABLE 10 % CD45+ recovery experiments. Cell suspension Sample immunogenicity Description % CD45+ Actual spike (tested in MLR (in % of cell cells (%, based on 60% and expressed types in the (detected CD45+ cells in this % Difference as IL-2R in mixture) by FACS) hPBMC batch) from actual spike pg/mL) 100% amnion 0.65 N/A N/A 20.23 0% PBMC N/A N/A N/A 15.6 (negative control) 100% PBMC 61.51 N/A N/A 86.31 (positive control) 20% PBMC + 4.26 12 64.5% 24.38 80% Amnion 10% PBMC + 2.24 6 62.7% 21.17 90% Amnion 5% PBMC + 1.7 3 43.3% 16.75 95% Amnion 2.5% PBMC + 1.36 1.5 Not 15.9  97.5% calculated* Amnion 1.25% PBMC + 1.06 0.75 Not 12.27 98.75% calculated* Amnion Notes: N/A—not applicable; *Not calculated - values are close to the method detection limits

Example 18 Protein Array Analyses

The protein profiles of amniotic and chorionic membranes were investigated using a SearchLight Multiplex chemiluminescent array. The presence of proteins in tissue membrane extracts and secreted by tissues in culture medium was investigated. For comparison, two commercially available products containing living cells, Apligraf and Dermagraft, were assayed.

Example 18.1. Dermagraft

Dermagraft membrane was thawed and washed according to the manufacturer's instructions. Dermagraft membrane was cut into 7.5 cm² pieces. For tissue lysates, one 7.5 cm² piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1.5 mL microcentrifuge tube and 500 μL of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No. 11836153001) was added and incubated on ice for 30 min with frequent vortexing. The sample was then centrifuged at 16000 g for 10 min. The supernatant was collected and sent for protein array analysis by Aushon Biosystems. For tissue culture, one 7.5 cm² piece of membrane was plated onto a well of a 12-well dish and 2 mL of DMEM+1% HSA+ antibiotic/antimycotic were added and incubated at 37° C.±2° C. for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems.

Example 18.2 Apligraf

Apligraf membrane was cut into 7.3 cm² pieces. For tissue lysates, one 7.3 cm² piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1.5 mL microcentrifuge tube and 500 μL of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No. 11836153001) was added and incubated on ice for 30 min with frequent vortexing. The sample was then centrifuged at 16000 g for 10 min. The supernatant was collected and sent for protein array analysis by Aushon Biosystems. For tissue culture, one 7.3 cm² piece of membrane was plated onto a well of a 12-well dish and 2 mL of DMEM+1% HSA+ antibiotic/antimycotic were added and incubated at 37° C.±2° C. for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems.

Example 18.3 Amniotic and Chorionic Membranes

Amniotic and chorionic membranes were isolated and packaged at −80° C.±5° C. according to the manufacturing protocols disclosed herein in Example 3. Packaged membranes were then thawed in a 37° C.±2° C. water bath and washed 3 times with DPBS. Membranes were cut into 8 cm² pieces. For tissue lysates, one 8 cm² piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1.5 mL microcentrifuge tube and 500 μL of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No. 11836153001) was added and incubated on ice for 30 min with frequent vortexing. Tissue lysate was then centrifuged at 16000 g for 10 min. The supernatant was collected and sent for protein array analysis by Aushon Biosystems. For tissue culture, one 8 cm² piece of membrane was plated onto a well of a 12-well dish and 2 mL of DMEM+1% HSA+ nantibiotic/antimycotic were added and incubated at 37° C.±2° C. for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems.

Initial testing consisted of an analysis of 36 proteins that are important for wound healing. The list of identified proteins is described in Table 11.

TABLE 11 List of selected proteins for analysis. Protein Group Based on Functionality Comments Metalloproteases Matrix Metalloproteinase 1 Matrix and growth factor (MMP1), MMP2,3,7,8,9,10,13 degradation; facilitate cell migration. MMP Inhibitors Tissue Inhibitors of MMPs Have angiogenic activity; (TIMP1 and 2) can be placed in the “angiogenic factors” group. Angiogenic Factors Angiotensin-2 (Ang-2); basic Majority of these factors Fibroblast Growth Factor also have growth and basic (bFGF); heparin-bound migration stimulatory Epidermal Growth Factor activities and can be (HB-EGF); EGF; FGF-7 (also placed in a group of known as Keratinocyte growth factors. Growth Factor-KGF); Platelet derived Growth Factors (PDGF) AA, AB, and BB; Vascular Endothelial Growth Factor (VEGF), VEGF-C and VEGF-D; Neutrophil gelatinase-associated lipocalin (NGAL); Hepatocyte Growth Factor (HGF); Placenta Growth Factor (PIGF); Pigment Epithelium Derived Factor (PEGF); Thrombopoetin (TPO) Protease Inhibitor/Protein Alpha-2-macroglobulin Inhibit protease activity; Carrier regulate growth factor activity. Growth Factors See “angiogenic factors” + See “angiogenic factors.” Transforming Growth Factor alpha (TGF-a) Cytokines Adiponectin (Acrp-30) Affect keratinocyte functions. Granulocyte Colony- Protection from infections. Stimulating Factor (G-CSF) Interleukin1 Receptor Regulate activity of Antagonist (IL-1RA) inflammatory cytokine IL- 1. Leukemia Inhibitory Factor Support angiogenic (LIF) growth factors. Chemokines SDF-1beta Attracts endothelial and other stem cells from circulation to wound site. Regulators of IGF Insulin-like growth factor Regulate IGF activity. binding protein (IGFBP1,2,3)

Example 18 Protein Expression in Present Placental Products

Preliminary protein array data analyses showed that the majority of selected testing factors (refer to Table 11) were expressed in amniotic membrane, chorionic membrane, Apligraf, and Dermagraft. Three proteins were identified as unique for the amniotic membrane and/or the chorionic membrane which are undetectable in Apligraf and Dermagraft. These proteins are EGF, IGFBP1, and Adiponectin. All three proteins are important for wound healing. FIG. 13 depicts expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic or chorionic membranes. AM75 and AM 78 are placental products of the present invention (e.g. cryopreserved), CM75 and CM78 are cryopreserved chorionic membrane products. These proteins are believed by the inventors to facilitate the therapeutic efficacy of the present placental products for wound healing.

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing EGF, IGFBP1, and/or adiponectin

Example 19 Wound Healing Proteins are Secreted for a Minimum of 14 Days

Placental products of the present invention demonstrate a durable effect, desirable for wound healing treatments. The extracellular matrix and presence of viable cells within the amniotic membrane described in this invention allow for a cocktail of proteins that are known to be important for wound healing to be present for at least 14 days. Amniotic membranes were thawed and plated onto tissue culture wells and incubated at 37° C.±2° C. for 3, 7, and 14 days. At each time point, a sample of the culture supernatant was collected and measured through protein array analysis as described in Example 18. Table 12 illustrates the level of various secreted factors in tissue culture supernatants from two donors of amniotic membranes at 3, 7 and 14 days as measured through protein array analysis.

TABLE 12 Levels of proteins secreted in amnion tissue culture supernatants at different time points (pg/ml). Day 3 Day 7 Day 14 hACRP30 548.03 766.73 371.56 hAlpha2Macroglobulin 69687.55 31764.00 48477.62 hANG2 0.00 9.28 1.65 hEGF 3.06 2.51 2.32 hbFGF 40.80 85.46 269.97 hFibronectin 1932101.25 3506662.00 6019286.50 hHBEGF 41.78 80.50 78.09 hHGF 5358.09 9327.67 18081.16 hIGFBP1 2654.57 6396.11 4666.88 hIGFBP2 4379.76 23797.46 21784.21 hIGFBP3 36030.52 107041.71 13350.99 hIL1ra 116593.20 675.09 4927.52 hKGF 7.29 13.86 36.59 hMMP1 323249.53 1727765.60 15272931.52 hMMP10 14804.44 20557.91 16194.56 hMMP13 92.92 408.17 399.01 hMMP2 38420.90 322500.72 3283119.13 hMMP3 66413.54 283513.74 3598175.53 hMMP7 128.51 147.65 4005.14 hMMP8 463.32 2109.21 2331.47 hMMP9 6139.53 25810.38 60483.67 hNGAL 15754.19 70419.63 721923.09 hPDGFAA 18.02 58.69 16.31 hPDGFAB 16.58 58.41 28.30 hPDGFBB 1.94 21.67 5.84 hPEDF 6793.74 21645.90 169990.84 hSDF1b 0.00 24.09 37.12 hTGFa 15.05 14.89 205.90 hTGFb1 334.07 341.53 680.33 hTGFb2 119.59 207.79 731.96 hTIMP1 197743.23 437492.21 247661.65 hTIMP2 4724.25 19970.76 189810.51 hTSP1 0.00 0.00 1274.62 hTSP2 13820.61 59695.21 991366.59 hVEGF 44.98 57.45 7.40 hVEGFC 548.03 766.73 371.56

Example 20 Interferon 2α (IFN-2α) and Transforming Growth Factor-β3 (TGF-β3)

Placental products described in this invention have been analyzed for the presence of IFN-2α and TGF-β3. Briefly, after thawing, the membranes were homogenized and centrifuged at 16,000 g to collect the resulting supernatants. Supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2α) and R&D Systems (TGF-β3). FIG. 14 shows significant expression of IFN-2α (A) and TGF-β3 (B) in placental product homogenates.

Without being bound by theory, interferon-2α and TGF-β3 may aid in the prevention of scar and contracture formation. IFN-2α may serve a role to decrease collagen and fibronectin synthesis and fibroblast-mediated wound contracture.

Example 21 Tissue Reparative Proteins in Amniotic Membranes

Placental product homogenates were analyzed for the presence of proteins that are important in tissue repair.

Placental product described in this invention have been analyzed for the presence of tissue reparative proteins. Briefly, the thawed products were incubated in DMEM+10% FBS for 72 hrs. The membranes were then homogenized in a bead homogenizer with the culture media. The homogenates were centrifuged, and the supernatants were analyzed on commercially available ELISA kits from R&D Systems. FIG. 15 shows significant expression of BMP-2, BMP-4, PLAB, PIGF, and IGF-1 in several donors of amniotic membranes.

Without being bound by theory, the inventors believe that efficacy of the present placental products for wound repair are due, in part, to the role of BMPs, IGF-1, and PIGF in the development and homeostasis of various tissues by regulating key cellular processes. BMP-2 and BMP-4 may stimulate differentiation of MSCs to osteoblasts in addition to promote cell growth; placental BMP or PLAB is a novel member of the BMP family that is suggested to mediate embryonic development. Insulin-like growth factor 1 (IGF-1) may promotes proliferation and differentiation of osteoprogenitor cells. Placental derived growth factor (PIGF) may acts as a mitogen for osteoblasts.

Example 22 MMPs and TIMPs

Both MMPs and TIMPs are among the factors that are important for wound healing. However, expression of these proteins must be highly regulated and coordinated. Excess of MMPs versus TIMPS is a marker of poor chronic wound healing. We investigated expression of MMPs and TIMPs and its ratio in amniotic membrane and chorionic membrane and compared it to the expression profile in Apligraf and Dermagraft.

Results showed that all membranes express MMPs and TIMPs; however, the ratio in the thawed placental products and chorionic membranes is significantly lower. Therefore, these membranes will be more beneficial for wound healing (FIG. 16).

Accumulated data indicate that the MMP to TIMP ratio is higher in cases of non-healing wounds. For example, the ratio between MMP-9 and TIMP1 is approximately 7-10 to one for good healing and 18-20 or higher for poor healing. Analysis of the ratio between MMPs and TIMPs secreted by placental tissues, Apligraf, and Dermagraft showed that the amniotic and chorionic membrane products contain MMPs and TIMPs at an approximate ratio of 7, which is favorable for wound healing. In contrast, Dermagraft had a ratio>20, and Apligraf had a ratio>200.

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing MMP-9 and TIMP1 at a ratio of about 7-10 to one.

Example 23 α2-Macroglobulin

α2-macroglobulin is known as a plasma protein that inactivates proteinases from all 4 mechanistic classes, serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases. Another important function of this protein is to serve as a reservoir for cytokines and growth factors, examples of which include TGF, PDGF, and FGF. In the chronic wounds like diabetic ulcers or venous ulcers, the presence of high amount of proteases leads to rapid degradation of growth factors and delays in wound healing. Thus, the presence of α2-macroglobulin in products designed for chronic wound healing will be beneficial. Results of the protein array analysis showed that amniotic and chorionic membranes contain α2-macroglobulin (Table 13). Although these preliminary data show high variability between donors, the importance of this protein in wound healing prompted the additional evaluation of α2-macroglobulin in placental tissues using a single analyte ELISA instead of protein array, which is a useful tool to evaluate the presence of multiple proteins in one sample for profiling.

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing α2-macroglobulin.

TABLE 13 Expression of α2-macroglobulin in placental tissue protein extracts. α2-macroglobulin Sample (pg/mL/cm²) AM75 7 CM75 790 AM78 53042 CM78 1014

Example 24 Establishment of EGF as a Marker for Amniotic Tissue Potency

EGF is among the factors that are important for wound healing (Schultz et al., 1991, Komarcevic, 2000, and Hong et al., 2006). The absence or decreased amount of EGF is one characteristic of chronic wounds (Harding et al., 2002). Evaluation of proteins derived from amniotic membrane samples prepared according to the developed manufacturing process disclosed by the present application reveal that EGF is one of the major factors secreted in higher quantities by these tissues. The importance of EGF for wound healing together with high levels of EGF detected in the presently disclosed amniotic membranes support selection of EGF as a potency marker for evaluation of membrane products manufactured for clinical use pursuant to the present disclosure. A commercially available ELISA kit from R&D Systems was selected for evaluation of its suitability to measure EGF secreted by amniotic membranes. ELISA qualification meets the standards established by the FDA and ICH guidances for bioanalytical assay validation (Validation of Analytical Procedures: Text and Methodology Q2(R1), 1994; ICH Harmonized Tripartite Guideline and Guidance for Industry Bioanalytical Method Validation, 2001). Amniotic membranes evaluated for expression of EGF by this method confirmed protein array data and further demonstrated that EGF was a unique factor expressed at clinically significant levels in these tissues.

Example 25 Amniotic Tissue Expression of EGF

Protein array analysis provided initial evidence that EGF was uniquely expressed in amniotic membranes but not in chorionic membranes (Table 14). The levels of EGF measured in amniotic membranes were of clinical significance.

TABLE 14 Protein array data showing range of expression of EGF in amniotic and chorionic membranes from multiple donors. Amnion Chorion (pg/ml) (pg/ml) EGF 127.3-361.4 0-0.8

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing EGF, optionally in substantial amounts.

Homogenate of Placental Products

Placental products were thawed until no remaining frozen cryomedia was present. Membranes were then removed from bags and cut into 4 cm×2 cm pieces while still adhered to nitrocellulose. Each piece of tissue was then removed from the nitrocellulose and washed twice with PBS. Each tissue was then snap frozen in a homogenization tube using liguid nitrogen. Subsequently, one pre-cooled 5 mm steel bead was added to each tube; samples were then homogenized using a Qiagen Tissue Lyser according to the manufacturer's recommendations in 500 μL homogenization media. Tissue homogenates were stored at −80° C.±5° C. until analyzed by ELISA for EGF expression.

ELISA Procedure and Validation

Samples were analyzed for the expression of EGF using the Quantikine Human EGF ELISA Kit (R&D Systems) according to the manufacturer's recommendations. Several parameters were tested to establish the test criteria and to show the suitability of this ELISA kit to measure EGF in placental tissue samples. Assay performance was assessed by analyzing linearity, range, lower and upper limits of quantitation (LLOQ and ULOQ), precision, accuracy, and robustness. Experimental data (Table 14) showed that the quantitation range of this assay was 3.9-250 pg/mL EGF. The intra- and inter-assay CVs ranged from 1.22 to 5.80% and 2.73 to 7.53%, respectively. Additionally, sample recovery analysis demonstrated accuracy within 20%. Furthermore, this assay showed dilutional linearity and specificity. Ruggedness was also demonstrated by assay insensitivity to variations introduced by different analysts.

TABLE 15 Established ELISA parameters for measuring EGF in placenta homogenates. Calibration Standard 3.9-250 pg/mL Range Assay Quantitation 7.8-250 pg/mL Range LLOQ 7.8 pg/mL LOD 2.18 pg/mL ULOQ 250 pg/mL

EGF Expression in Amniotic Membranes

Measurement of EGF in amniotic preparations has proven to be both reliable and reproducible. Measurement of EGF in multiple donors showed that this method of quantification was a valuable means of evaluating potency in tissue prepared pursuant to the present disclosure for use in a clinical setting. FIG. 17 shows representative expression of EGF in a thawed placental product and chorionic membrane prepared and analyzed by the methods described above. Results have been reproduced in multiple tissue preparations.

These data are consistent with certain embodiments of the present invention that provide a placental product comprising an amniotic membrane containing EGF.

Example 26 Placental Tissues Enhance Cell Migration and Wound Healing

The process of wound healing is highly complex and involves a series of structured events controlled by growth factors (Goldman, 2004). These events include increased vascularization, infiltration by inflammatory immune cells, and increases in cell proliferation. The beginning stages of wound healing revolve around the ability of individual cells to polarize towards the wound and migrate into the wounded area in order to close the wound area and rebuild the surrounding tissue. Upon proper stimulation, several different types of cells including epithelial, endothelial, mesenchymal, and fibroblastic cells are implicated in the wound healing process (Pastar et al, 2008 and Bannasch et al., 2000). Specifically, they proliferate and migrate into the wound area to promote healing. Therefore, experiments were conducted to determine if factors secreted from amniotic and chorionic membranes produced pursuant to the present disclosure promote cell migration and wound field closure. To accomplish this, a commercially available wound healing assay (Cell Biolabs) and a highly accepted human microvascular endothelial cell line (HMVEC, Lonza Inc.) were utilized. Results indicated that cell migration was enhanced by treatment with conditioned media from the placental membranes.

In Vitro Cell Migration Assay

Human microvascular endothelial cells (HMVECs) were grown under normal cell culture conditions in defined complete media (Lonza Inc.). To assess migration and wound field closure, a commercially available wound healing assay was used (Cell Biolab).

FIG. 18 depicts the Cell Biolabs 24-well Cytoselect wound healing assay. (Figure reproduced from Cell Biolabs).

Cells were collected via trypsinization, pelleted, and counted before being resuspended in complete media at a density of 2×10⁵ cells/mL. 250 μL (5×10⁴ cells) cell suspension was then pipetted into each side of a well containing a wound healing insert (Cytoselect 24-well Wound Healing Assay Plate, Cell Biolabs). The cells were grown for 24 hours in complete media. After 24 hours, the wound inserts were removed. At the same time, complete media was removed and replaced with experimental media. Complete media and basal media were used as positive and negative controls, respectively. To generate experimental media, placental membranes were incubated for 3 days in DMEM with 1% human serum albumin (HSA) in a tissue culture incubator. The resulting tissue and media were then placed in eppendorf tubes and spun at high speed in a microcentrifuge. The supernatants were collected and stored at −80° C.±2° C. until use. For migration and wound healing studies, conditioned media from placental membranes was diluted 1:20 in basal media before being added to experimental wells. After 18 hours, the media was removed, and the cells were fixed for 20 min in 4% paraformaldehyde and stained with crystal violet. The wound field in each well was then photographed. Wound healing was determined by the amount of wound field still visible at the end of the experiment when compared to control pictures taken before conditioned media was added to the wells.

Placental Membrane Conditioned Media Supports Cell Migration and Wound Field Closure

Conditioned media from amniotic and chorionic membranes was used to assess the potential for these membranes to promote cell migration and wound field closure. Conditioned media from placental amniotic, chorionic, and a combination of amniotic/chorionic membranes supported migration of cells into the experimental wound field.

FIG. 19 depicts representative images of cells treated with 5% conditioned media from amniotic, chorionic, or a combination of amniotic/chorionic tissue as well as positive and negative controls. Wound field is 0.9 mm in width.

The ability of factors from placental membranes produced pursuant to the present disclosure to promote HMVEC migration indicated that these tissues have the ability to enhance wound healing. Additionally, based on the insight of the inventors, it has been surprisingly discovered that these tissues also enhance revascularization since the HMVEC cell line is derived from vascular endothelial cells.

These data demonstrate that placental products of the present invention produce unexpectedly superior levels of factors that promote wound healing therapies.

Example 27 Biochemical Profile of the Supernatants from Exemplary Placental Tissue Products

Table 16 depicts the biochemical profile of the supernatants of examplary placental products of the invention (results adjusted per cm² after subtraction of the negative background).

TABLE 16 Factors in Placental Tissue Product (pg/cm²). Units Apligraf Dermagraft AM75 CM75 AM78 CM78 hMMP1 pg/ml/cm² 1964945.37 14818.20 2821.85 3531.81 117326.89 95.46 hMMP7 pg/ml/cm² 911.54 0.00 0.00 0.00 3.96 0.00 hMMP10 pg/ml/cm² 0.00 0.00 113.94 0.00 0.00 0.00 hMMP13 pg/ml/cm² 21.61 0.00 0.00 0.00 0.71 0.00 hMMP3 pg/ml/cm² 208281.70 180721.52 170.26 161.52 8325.17 0.00 hMMP9 pg/ml/cm² 8872.28 19321.39 214.78 1455.11 630.56 57.59 hMMP2 pg/ml/cm² 153341.77 19712.21 287.14 37.93 3823.38 24.44 hMMP8 pg/ml/cm² 36.92 12.19 0.00 0.00 0.00 0.00 hTIMP1 pg/ml/cm² 2487.18 10909.84 569.23 883.05 28743.48 97.94 hTIMP2 pg/ml/cm² 7285.53 1796.56 89.29 13.72 424.06 4.83 MMP/TIMP 239.26 19.72 6.81 6.26 4.50 2.62

Example 28 Biochemical Profile of the Lysates from Exemplary Placental Tissue Products

Table 17 depicts the biochemical profile of the lysates of examplary placental tissue products of the invention (results adjusted per cm² after subtraction of the negative background).

TABLE 17 AM75 lysate AM78 lysate CM75 lysate CM78 lysate pg/ml pg/ml pg/ml pg/ml hACRP30 50.8 1154.6 1213.7 225.3 hAlpha2- 1910.6 426191.6 8174.4 9968.6 Macroglobulin hEGF 127.3 361.4 0.0 0.8 hbFGF 119.1 821.5 375.0 351.3 hGCSF 0.7 3.2 1.2 0.7 hHBEGF 127.5 168.0 15.4 84.5 hHGF 3943.7 15060.0 29979.6 50392.8 hIGFBP1 5065.0 9456.6 934.0 1443.6 hIGFBP2 12460.8 5569.7 135.9 134.6 hIGFBP3 50115.7 41551.4 4571.5 11970.2 hIL1ra 3881.0 32296.9 5168.2 525.5 hKGF 1.4 8.8 3.1 1.5 hLIF 0.0 4.2 0.0 0.0 hMMP1 9144.1 20641.2 2882.9 6582.3 hMMP10 0.0 15.5 79.3 87.5 hMMP2 2067.3 4061.9 949.5 748.8 hMMP3 0.0 36.2 0.0 0.0 hMMP7 5.1 11.4 4.5 9.1 hMMP8 0.0 0.0 0.0 0.0 hMMP9 92.2 2878.1 2676.2 1259.3 hNGAL 6900.1 6175.9 938.5 229.7 hPDGFAA 0.0 12.5 39.8 35.2 hPDGFAB 11.2 31.3 14.4 14.0 hPDGFbb 4.6 13.4 4.0 1.3 hPEDF 0.0 652.6 0.0 0.0 hTIMP1 7958.1 35955.6 50712.3 17419.9 hTIMP2 3821.8 7443.2 640.7 780.0 hVEGF 3.3 11.8 125.2 8.4 hVEGFC 46.5 150.0 123.5 51.7 hVEGFD 25.7 31.0 15.0 20.4

Example 29 Use of Placental Products for Treating Diabetic Foot Ulcers

Purpose: Despite bioengineered skin substitutes that contain human fibroblasts or a combination of human fibroblasts and keratinocytes, published rates of chronic wound healing remain low, with approximately half of all wounds recalcitrant to even these newer therapies. Morbidity and mortality from diabetic foot ulceration are substantial as the 5 year mortality rate following a lower extremity amputation is between 39% and 68%. (Page J. J of Foot & Ankle Surgery 2002; 41(4):251-259; Isumi Y., et al. Diabetes Res and Clin Practice 2009; 83:126-131).

A instant membrane product, which provides necessary angiogenic and anti-inflammatory growth factors was introduced in an effort to improve outcomes of patients with chronic skin ulceration at-risk for amputation.

Objective: Patients with chronic diabetic foot ulceration, unresponsive to available therapy and at-risk for amputation were considered for treatment. All wounds were aggressively debrided prior to graft application. Patients were evaluated regularly and application of a membrane product of the present invention was at the discretion of the treating physician. Offloading was encouraged in both patients.

Introduction

According to the United States Food and Drug Administration (FDA), a chronic, cutaneous ulcer is defined as a wound that has failed to proceed through an orderly and timely series of events to produce a durable structural, functional and cosmetic closure(2). The most common chronic wounds include pressure ulcers and leg ulcers. The triad of peripheral neuropathy, deformity, and minor trauma has emerged as the most frequent causes of insult that lead to foot ulcerations. In terms of healing rates, an appropriate benchmark for a chronic wound is a decrease of 10% to 15% in size every week, or 50% decrease in size over a one-month period. A three-year retrospective cohort study performed by Ramsey et al. of 8,905 patients in a large health maintenance organization who have diabetes reported a 5.8% cumulative incidence of ulceration. At the time of diagnosis, 15% of these patients developed osteomyelitis and 16% required partial amputation of a lower limb.

Approximately 80% to 85% of lower extremity amputations are preceded by foot ulcerations. Morbidity and mortality from diabetic foot ulceration are substantial as the 5 year mortality rate following a lower extremity amputation is between 39% and 68%. (2). These mortality rates are higher than the five-year mortality rates for breast cancer, colon cancer, and prostate cancer.

Despite all of the advances in bioengineered tissue for the treatment of chronic diabetic ulcerations, there are an abundance of patients whose ulcerations are resistant to therapy, and result in a chronic wound. Because of healing rates that only approach 50% with these newer therapies, the use of stem cells in regenerative medicine has been of particular interest recently. The ultimate aim is to promote restoration of functional skin. A preliminary study was performed by Fiami et al. in which they isolated mesenchymal stem cells from umbilical cord blood and inoculated them onto a piece of de-epithelialized dermis. The results of this preliminary study showed that peripheral stem cells are capable of surviving and expressing neoangiogenesis. In addition to showing promise for tissue repair, mesenchymal stem cells exhibit low immunogenicity and can be transplanted universally without having to undergo compatibility testing between the donor and recipient.

In this study, clinical evidence of remarkable healing using an instant membrane product for the treatment of two chronic wounds that amputation was considered. The fundamentals of wound management are still the cornerstone of comprehensive wound care in any treatment protocol including adequate debridement, offloading, maintaining a moist environment, and adequate perfusion and infection control.

Materials

An instant membrane product was made as taught herein, comprising an allograft derived from the aminon comprising a bilayer of native epithelial cells on a basement membrane and a stoma layer consisting of neonatal fibroblasts, extracellular matrix (ECM) and mesenchymal stem cells (MSC).

Limb Salvage: Case One History and Physical Examination

A 70 year old male presented to the emergency department with bulla formation on the dorsolateral aspect of his right foot between the fourth and fifth digits, edema and pain, and a small lesion lateral to the fifth digit. The patient reported a history of minor trauma to the area two weeks prior to presentation. The patient had a history of type II diabetes mellitus, hypertension, heart failure, chronic obstructive pulmonary disease, and chronic kidney disease treated with hemodialysis three times a week. The patient had a surgical history of an aorta-venous graft replacement. He denied any history of alcohol, tobacco or drug use. Physical exam revealed no active purulent drainage or malodor, and no tenderness on palpation. The vascular exam revealed non-palpable pulses in the dorsalis pedis and posterior tibial arteries. Doppler exam revealed a monophasic dorsalis pedis pulse with a biphasic posterior tibial artery pulse. The fifth digit had gangrenous changes and was cold on palpation. There were ischemic changes of the fourth digit. Radiographic evaluation revealed scattered air densities indicative of soft tissue gas in the fourth interspace as well as the tip of the fifth digit.

Preoperative Management

The patient was started on intravenous antibiotics of vancomycin and piperacillin and tazobactam at appropriate renal dosing.

Operative Management

The patient was taken to the operating room where an incision and drainage of the fourth interspace was performed, and a partial fifth ray amputation to the level of the metatarsal head was performed without complication. The wound was left open and packed with sterile gauze moistened with sterile normal saline, and covered with a sterile compressive dressing. Intraoperative findings revealed liquefactive necrosis of surrounding tissues with purulence and malodor. The patient underwent 2 subsequent surgical debridements, with the second resulting in further removal of the fourth and fifth metatarsal shafts. In a third surgery further debridement of necrotic soft tissue and amputation of the fourth digit was performed. On May 20, 2010 treatment with an instant membrane product was initiated. Prior to the graft placement the patient had undergone successful recanalization of the popliteal artery and the peroneal artery without significant residual stenosis.

Postoperative Course

The patient followed up with his podiatric surgeon within 2 days of being discharged from the hospital. Upon initial exam, there were no clinical signs of infection, and the proximal dorsal incision appeared coapted. The third digit was dusky and cool in appearance. Radiographs were taken which showed no evidence of soft tissue gas or acute osteomyelitis. A dry sterile dressing was applied. The patient received applications of the insant membrane product at 6 additional visits in an outpatient office. Prior to each application the wound was evaluated for abscess, cellulitis, drainage, hematoma formation, and infection. At each visit, the wound decreased in size and appeared more granular in nature as compared to previous visits. At the time of the third application the wound had decreased in size 50%.

At 19 weeks the wound was considered closed, and the patient was instructed to remain weight bearing on the affected limb with the use of a surgical shoe only.

Photographs of the remarkable wound healing mediated by a placental product of the present invention as shown in FIG. 20. Panel A: First application of an instant membrane product; B: 8 weeks post first an instant membrane product application; C: 10 ½ weeks post first an instant membrane product application; D: 12 weeks post first an instant membrane product application; E: 19 weeks post first an instant membrane product application.

Limb Salvage: Case Two History and Physical Examination

A 44-year old male presented to an outpatient office with a large ulceration on the plantar aspect of his left hallux, secondary to a previous trauma a few weeks prior to the visit. The patient had a history of diabetes mellitus for the past five years complicated by peripheral neuropathy, hypertension, dyslipidemia, and osteomyelitis. Past surgical history included abdominal aortic aneurysm repair and circumcision. On physical exam the ulceration measured 4.0 cm×2.0 cm×1.5 cm, probing to the distal phalanx with exposed tendon. There was no ascending cellulitis or lymphangitis, and no increased temperature gradient. Capillary fill time, hair growth, and tissue turgor were all normal. There were palpable pulses in the dorsalis pedis and the posterior tibial artery. Radiographic exam was negative for soft tissue gas. Magnetic resonance imaging revealed osteomyelitis in the distal aspect of the proximal phalanx and the distal phalanx of the great toe with a small soft tissue abscess in the region of the dorsal soft tissue adjacent to the distal phalanx.

Preoperative Management

The patient was started on intravenous antibiotics. He was taken to the operating room for excisional debridement of all nonviable tissue and application of the instant membrane product.

Operative Management

The ulceration was debrided to healthy tissue with utilization of both sharp dissection and Versajet™, leaving the head of the proximal phalanx exposed plantarly. The instant membrane product was then placed over the wound bed and exposed bone. The patient tolerated the procedure without complication. The patient was discharged from the hospital the day after surgery on a five week course of intravenous antibiotic therapy.

Postoperative Course

The patient was instructed to remain strictly non-weight bearing to the affected limb, and returned for follow-up on post-operative day 6. The dressing was clean, dry and intact. There were no post-operative complications such as abscess, cellulitis, discomfort, or drainage and no clinical signs of infection. The patient received a total of 7 stem cell graft applications over the course of the next 8 weeks. At each visit the wound was inspected for clinical signs of infection. Evaluation at each visit revealed marked development in granulation tissue to the wound base and significant decrease in size. Eight weeks after the initial application of the allograft tissue the wound was closed.

Photographs of the remarkable wound healing mediated by a placental product of the present invention as shown in FIG. 21. Panel A: Osteomyelitis, tendon exposed, probed to bone. First stem cell graft was applied after surgical debridement; B: Status post 1 application of stem cell graft, wound is granular in nature and no signs of infection; C: 3 weeks post surgical intervention; 2 applications on the instant membrane product, the wound is considerably smaller in circumference and depth; D: 6 weeks post surgical intervention the wound is almost closed; E: 8 weeks and 7 applications of the instant membrane product, the wound is closed.

Conclusion

Despite the tremendous progress in skin tissue engineering in the past few decades, current therapy has limited efficacy in the treatment of chronic diabetic ulceration. As shown in this case report of two patients, the use of advanced therapies containing stem cells may prove useful to ultimately heal these patients in lieu of amputation, reduce mortality rates, and at the same time be a cost effective alternative to standard treatments currently on the market. Both patients highlighted in this case report received 7 applications of a membrane product of the present invention. Complete healing occurred in both patients. There were no reported complications associated with treatment; the instant membrane product was safe and effective in an initial evaluation of two patients with diabetic foot ulceration at-risk for amputation. These results indicate that patients with recalcitrant, chronic wounds should be considered for this novel therapy. 

1. A placental product comprising: a amniotic membrane, wherein the amniotic membrane: a. comprises viable therapeutic cells; and b. is substantially free of functional CD14+ macrophages; and optionally, wherein the viable therapeutic cells are native to the amniotic membrane.
 2. The placental product of claim 1, wherein the amniotic membrane is substantially free of vascularized tissue or vascularized tissue-derived immunogenic cells.
 3. The placental product of claim 1, wherein the amniotic membrane comprises a layer of epithelial cells.
 4. The placental product of claim 1, wherein the amniotic membrane comprises one, two, or three layers selected from: a compact layer, a layer of fibroblast, and a spongy layer.
 5. The placental product of claim 1, wherein the amniotic membrane is substantially free of immunogenic maternal cells.
 6. The placental product of claim 1, wherein the amniotic membrane is substantially free of maternal dendritic cells.
 7. The placental product of claim 1, wherein the amniotic membrane is substantially free of maternal leukocytes.
 8. The placental product of claim 1, wherein the viable therapeutic cells comprise one or more of: a. MSCs; b. fibroblasts; c. epithelial cells; d. stromal cells; and e. stromal cells and epithelial cells.
 9. The placental product of claim 8, wherein the amniotic membrane is substantially free of vascularized tissue or vascularized tissue-derived immunogenic cells.
 10. The placental product of claim 9, wherein the amniotic membrane comprises a layer of epithelial cells.
 11. The placental product of claim 9, wherein the amniotic membrane comprises one, two, or three layers selected from: a compact layer, a layer of fibroblast, and a spongy layer.
 12. The placental product of claim 8, wherein the amniotic membrane is substantially free of immunogenic maternal cells.
 13. The placental product of claim 8, wherein the amniotic membrane is substantially free of maternal dendritic cells.
 14. The placental product of claim 8, wherein the amniotic membrane is substantially free of maternal leukocytes.
 15. The placental product of any of claims 8-14, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable.
 16. The placental product of claim 15, wherein the stromal cells are about 2,000 to about 15,000 in number per cm² of the amniotic membrane.
 17. The placental product of any of claims 15, wherein the stromal cells are at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 in number per cm².
 18. The placental product of any of claims 8-14, wherein about 40% to about 92.5% of the stromal cells are viable.
 19. The placental product of claim 18, wherein the stromal cells are about 2,000 to about 15,000 in number per cm² of the amniotic membrane.
 20. The placental product of any of claims 19, wherein the stromal cells are at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 in number per cm².
 21. The placental product of any of claims 1-14, wherein the amniotic membrane comprises about 2,000 to about 15,000 stromal cells per cm² of the amniotic membrane, optionally, wherein the stromal cells are native to the amniotic membrane.
 22. The placental product of claim 21, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable.
 23. The placental product of claim 21, wherein about 40% to about 92.5% of the stromal cells are viable.
 24. The placental product of any of claims 1-14, wherein the amniotic membrane comprises at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 stromal cells per cm² of the amniotic membrane, optionally, wherein the stromal cells are native to the amniotic membrane.
 25. The placental product of claim 24, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable.
 26. The placental product of claim 24, wherein about 40% to about 92.5% of the stromal cells are viable.
 27. The placental product of any claims 1-14, wherein the amniotic membrane comprises about fibroblasts in about 10% to about 30% fibroblasts per total cells.
 28. The placental product of any of claims 1-14, wherein the amniotic membrane comprises fibroblasts in about 2% to about 30% fibroblasts per total cells.
 29. The placental product of any of claims 1-28, wherein the placental product or amniotic membrane secretes between about 0 to about 420 pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm piece of the placental product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
 30. The placental product of claim 29, wherein said about between about 0 to about 420 pg/mL TNF-α is: about 0 to about 350 pg/mL TNF-α or 0 to about 280 pg/mL TNF-α.
 31. The placental product of any of claims 1-14, wherein the amniotic membrane further comprises one or more native factors consisting of the group of IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9 and TIMP1; wherein natives factors are present in an amount: i. substantially greater per gram of amniotic membrane than that of a fresh unprocessed amniotic membrane; ii. substantially greater per native amniotic membrane cell than that of a fresh unprocessed amniotic membrane; or iii. substantially greater per μm of amniotic membrane.
 32. The placental product of any of claims 1-14, wherein the amniotic membrane has a thickness of about 20 μm to about 50 μm.
 33. The placental product of any of claims 1-14, wherein the amniotic membrane comprises a basement membrane.
 34. The placental product of any of claims 1-14, further comprising a chorionic membrane, optionally wherein the chorionic membrane is substantially free of trophoblasts.
 35. A placental product comprising: a cryopreservation medium and an amniotic membrane, wherein: a. the amniotic membrane is substantially free of functional CD14+ macrophages; and b. the cryopreservation medium comprises a cryopreserving amount of a cryopreservative; and optionally, wherein the amniotic membrane is substantially free of vascularized tissue or vascularized tissue-derived immunogenic cells.
 36. The placental product of claim 35, wherein the amniotic membrane comprises viable therapeutic cells, optionally, wherein the viable therapeutic cells are native to the amniotic membrane and of one or more of the types: a. MSCs; b. fibroblasts; c. epithelial cells; d. stromal cells and e. stromal cells and epithelial cells.
 37. The placental product of claim 36, wherein the cryopreservative comprises a cell-permeating cryopreservative, a non-cell-permeating cryopreservative, or a combination thereof, optionally wherein the permeating cryopreservative comprises: a. DMSO; or b. DMSO in a majority amount.
 38. The placental product of claim 37, wherein the cell-permeating cryopreservative: a. does not comprise glycerol in a majority amount; or b. does not comprises a substantial amount of glycerol.
 39. The placental product of claim 37, wherein the cell-permeating cryopreservative comprises a DMSO in amount of: about 2% to about 20%, about 5% to about 20%, about 5% to about 15%, or about 7% to about 13%.
 40. The placental product of claim 37, wherein the cryopreservation medium is present in an amount of at least: about 20 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, or about 50 ml.
 41. The placental product of claim 39, wherein the cryopreservation medium is present in an amount of at least: about 20 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, or about 50 ml.
 42. The placental product of claim 37, wherein the cryopreservation medium further comprises albumin, optionally wherein the albumin is HSA.
 43. The placental product of claim 39, wherein the cryopreservation medium further comprises albumin, optionally wherein the albumin is HSA.
 44. The placental product of any of claims 36-43, wherein the cryopreservative comprises a non-cell-permeating cryopreservative selected from is a polyvinylpyrrolidone, a starch, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
 45. The placental product of any of claims 36-43, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 46. The placental product of claim 45, wherein the stromal cells are about 2,000 to about 15,000 in number per cm² of the amniotic membrane.
 47. The placental product of claim 45, wherein the stromal cells are at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 in number per cm².
 48. The placental product of any of claims 36-43, wherein about 40% to about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 49. The placental product of claim 48, wherein the stromal cells are about 2,000 to about 15,000 in number per cm² of the amniotic membrane.
 50. The placental product of any of claims 48, wherein the stromal cells are at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 in number per cm².
 51. The placental product of any of claims 35-43, wherein the amniotic membrane comprises about 5,000 to about 50,000 stromal cells per cm² of the amniotic membrane, optionally, wherein the stromal cells are native to the amniotic membrane.
 52. The placental product of claim 51, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 53. The placental product of claim 51, wherein about 40% to about 92.5% of the stromal cells are viable.
 54. The placental product of any of claims 35-43, wherein the amniotic membrane comprises at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 stromal cells per cm² of the amniotic membrane, optionally, wherein the stromal cells are native to the amniotic membrane.
 55. The placental product of claim 54, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 56. The placental product of claim 54, wherein about 40% to about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 57. The placental product of any claims 35-43, wherein the amniotic membrane comprises fibroblasts in about 2% to about 30% fibroblasts per total cells.
 58. The placental product of any of claims 35-43, wherein the amniotic membrane comprises CD14+ macrophage at less than about 10% or less than about 2% or less than about 0.5% of the total cells.
 59. The placental product of any of claims 35-43, wherein the amniotic membrane is substantially free of: a. maternal dendritic cells, maternal leukocytes, or a combination thereof, or b. immunogenic maternal cells,
 60. The placental product of any of claims 35-43, wherein the product comprises about 2,000 to about 15,000 stromal cells per cm² and wherein the viability of said stromal cells after a freeze-thaw cycle is about 70% to about 95% relative to fresh placental or amniotic membrane.
 61. The placental product of any of claims 35-43, wherein after a freeze-thaw cycle, the placental product or amniotic membrane secretes between about 0 to about 420 pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm piece of the placental product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
 62. The placental product of any of claims 35-43, wherein after a refrigeration-freeze-thaw cycle, the placental product or amniotic membrane secretes between about 0 to about 420 pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm piece of the placental product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
 63. The placental product of claim 61, wherein said about between about 0 to about 420 pg/mL TNF-α is: about 0 to about 350 pg/mL TNF-α or 0 to about 280 pg/mL TNF-α.
 64. The placental product of any of claims 35-43, wherein the amniotic membrane further comprises one or more native factors selected from the group consisting of IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9 and TIMP1; wherein the one or more native factors are present in an amount, wherein the amount is: i. substantially greater per gram of amniotic membrane than that of a fresh unprocessed amniotic membrane; ii. substantially greater per native amniotic membrane cell than that of a fresh unprocessed amniotic membrane; or iii. substantially greater per μm of amniotic membrane.
 65. The placental product of any of claims 35-43, wherein the amniotic membrane has a thickness of about 20 μm to about 50 μm.
 66. The placental product of any of claims 35-43, further comprising a chorionic membrane, optionally wherein the chorionic membrane is substantially free of trophoblasts.
 67. The placental product of claim 66, wherein the amniotic membrane and the chorion membrane are associated to each another in the native architecture.
 68. The placental product of claim 66, wherein the amniotic membrane and the chorionic membrane are not attached to one another in the native architecture.
 69. The placental product of any of claims 35-43, wherein the amniotic membrane comprises a layer of epithelial cells.
 70. The placental product of any of claims 35-43, wherein the amniotic membrane comprises one, two, or three layers selected from: a compact layer, a layer of fibroblast, and a spongy layer.
 71. The placental product of any of claims 35-43, wherein the amniotic membrane has a thickness of about 20 μm to about 50 μm.
 72. The placental product of any of claims 35-43, wherein the amniotic membrane comprises a layer of epithelial cells.
 73. The placental product of any of claims 35-43, wherein the amniotic membrane comprises a basement membrane.
 74. A placental product comprising: a cryopreservation medium and an amniotic membrane, wherein: a. the amniotic membrane: i. comprises a compact layer, layer of fibroblast, and a spongy layer, wherein each of said layers comprises viable therapeutic cells; and ii. is substantially free of vascularaized tissue and functional CD14+ macrophages; and b. the cryopreservation medium comprises a cryopreserving amount of a cryopreservative.
 75. The placental product of claim 74, wherein the amniotic membrane comprises about 2,000 to about 15,000 stromal cells per cm² of the amniotic membrane.
 76. The placental product of claim 75, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable.
 77. The placental product of any claim 75, wherein about 40% to about 92.5% of the stromal cells are viable.
 78. The placental product of claim 74, wherein the amniotic membrane comprises at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 stromal cells per cm² of the amniotic membrane.
 79. The placental product of claim 75, wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 80. The placental product of claim 75, wherein about 40% to about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
 81. The placental product of any of claims 74-80, wherein the amniotic membrane further comprises a basement membrane.
 82. The placental product of any of claims 74-81, wherein the amniotic membrane has a thickness of about 20 μm to about 50 μm.
 83. The placental product of any of claims 74-82, wherein the cryopreservative comprises DMSO.
 84. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises MSCs in an amount of: at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, about 1% to about 10%, or about 3% to about 10%, relative to the total number of cells in the amniotic membrane.
 85. The placental product of claim 84, wherein at least about 40%, about 50%, about 60%, or about 70% of the MSCs are viable.
 86. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises fibroblasts in an amount of: about 1%, about 20%, about 5% to about 15%, at least about 1%, at least about 2%, at least about 3%, or at least about 4 relative to the total number of cells in the amniotic membrane.
 87. The placental product of claim 86, wherein at least: about 40%, about 50%, about 60%, or about 70% of the fibroblasts are viable.
 88. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises stromal cells in an amount of: about 5% to about 40%, about 5% to about 30%, about 10% to about 30%, about 15% to about 25%, at least about 5%, at least about 10%, or at least about 15%, relative to the total number of cells in the amniotic membrane.
 89. The placental product of claim 88, wherein at least: about 40%, about 50%, about 60%, or about 70% of the stromal cells are viable after a freeze-thaw cycle
 90. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises epithelial cells in an amount of: about 60% to about 90%, about 70% to about 90%, about 40% to about 90%, about 50% to about 90%, at least about 40%, at least about 50%, least about 60%, or at least about 70%, relative to the total number of cells in the amniotic membrane.
 91. The placental product of claim 90, wherein at least: about 40%, about 50%, about 60%, or about 70% of the epithelial cells are viable after a freeze-thaw cycle.
 92. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises MSCs and functional macrophages in a ratio of greater than about any of: 5:1, 7:1, or 10:1.
 93. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises fibroblasts and functional macrophages in a ratio of greater than about any of: 10:1, 15:1, 20:1, or 25:1.
 94. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises fibroblasts and MSCs in a ratio of: about 4:1 to about 1:1 or about 3:1 to about 3:2, or about 2:1.
 95. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises MSCs in an amount of: at least about 1,000 cells/cm²′ at least about 2,000 cells/cm², about 1,000 to about 5,000 cells/cm², or about 2,000 to about 5,000 cells/cm².
 96. The placental product of claim 95, wherein at least: about 40%, about 50%, about 60%, or about 70% of the MSCs are viable after a freeze-thaw cycle
 97. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises fibroblasts in an amount of: at least about 2,000 cells/cm², at least about 4,000 cells/cm², about 2,000 to about 9,000 cells/cm² , or about 2,000 to about 9,000 cells/cm².
 98. The placental product of claim 97, wherein at least: about 40%, about 50%, about 60%, or about 70% of the fibroblasts are viable after a freeze-thaw cycle
 99. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises stromal cells in an amount of: at least about 4,000, at least about 8,000 cells/cm², about 4,000 to about 18,000 cells/cm², or about 4,000 to about 18,000 cells/cm².
 100. The placental product of claim 99, wherein at least: about 40%, about 50%, about 60%, or about 70% of the stromal cells are viable after a freeze-thaw cycle.
 101. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises epithelial cells in an amount of: at least about 10,000 cells/cm², at least about 20,000 cells/cm², at least about 32,000 cells/cm², about 10,000 to about 72,000 cells/cm² , about 20,000 to about 72,000 cells/cm², or about 32,000 to about 72,000 cells/cm²
 102. The placental product of claim 101, wherein at least: about 40%, about 50%, about 60%, or about 70% of the epithelial cells are viable after a freeze-thaw cycle.
 103. The placental product of any of claims 1-14, 35-43, or 74-80, wherein the amniotic membrane comprises functional macrophages in an amount of: less than about 3,000 cells/cm², less than about 1,000 cells/cm², or less than about 500 cells/cm².
 104. A method of treating a wound of a subject comprising administering the placental product of any of the preceding Claims to the wound.
 105. The method of claim 94, wherein the wound is an ulcer.
 106. The method of claim 95, wherein the ulcer is a foot ulcer, optionally a diabetic foot ulcer.
 107. The method of claim 95, wherein the ulcer is a diabetic foot ulcer.
 108. The method of any of claims 93-97, wherein the subject has diabetes.
 109. A method of ligament or tendon surgery comprising applying the placental product of any of claims 1-103 to a ligament or tendon.
 110. The method of claim 109, wherein the ligament or tendon surgery is a grafting surgery, a reconstruction surgery, or a repair of a torn ligament or tendon.
 111. The method of claim 110, wherein the grafting surgery comprises the attachment of a graft to a biological substrate, optionally, wherein the biological substrate is bone.
 112. The method of claim 111, wherein the graft is an allograft, a xenograft, or an autologous graft.
 113. The method of claim 110, wherein the ligament or tendon surgery is selected from: knee surgery, shoulder surgery, leg surgery, and arm surgery, elbow surgery, finger surgery, hand surgery, wrist surgery, toe surgery, foot surgery, ankle surgery.
 114. The method of claim 110, wherein the tendon is hamstring tendon, a bicep tendon, an Achilles Tendon, an extensor tendon, or a rotator cuff tendon.
 115. The method of claim 110, wherein the step of applying the placental product comprises: suturing the placental product to the tendon or ligament, enveloping the tendon or ligament with the placental product, or a combination thereof.
 116. A method of tissue grafting comprising applying the placental product of any of claims 1-103 to a tissue graft, and attaching the tissue graft to a biological substrate.
 117. The method of claim 116, wherein the placental product is applied to a portion of the tissue graft which is attached to the biological substrate.
 118. The method of claim 117, wherein the step of applying the placental product comprises: suturing the placental product to the graft, enveloping graft with the placental product, or a combination thereof.
 119. The method of claim 118, wherein the tissue graft comprises a tissue selected from: skin, cartilage, ligament, tendon, periosteum, perichondrium, synovium, fascia, and mesenter.
 120. A method of inhibiting fibrosis or adhesion at a wound site comprising applying the placental product of any of claims 1-103 to the wound site.
 121. The method of claim 120, wherein the wound site is surgery induced or associated with surgery.
 122. The method of claim 121 wherein the surgery is a surgery selected from: spine, laminectomy, knee, shoulder, or child birth, eye, cardiovascular, angiogenesis stimulation, burn, and brain/neurological procedures.
 123. The method of claim 120, wherein the wound site is a trauma related wound.
 124. A method of treating a tissue injury of a subject comprising administering the placental product of any of claims 1-103 to the tissue injury.
 125. The method of claim 124, wherein the tissue injury is an ulcer.
 126. The method of claim 125, wherein the ulcer is a foot ulcer, optionally a diabetic foot ulcer.
 127. The method of claim 126, wherein the ulcer is a diabetic foot ulcer.
 128. The method of any of claims 125 wherein the subject has diabetes.
 129. The method of any of claims 125 wherein the wound is associated with ischemia.
 130. A method of promoting migration or proliferation of at least one cell selected from the group consisting of fibroblasts, epithelial cells, and endothelial cells] in a subject comprising administering the placental product of any of claims 1-103 a subject in need thereof.
 131. A method of reorganizing extracellular matrix of a subject comprising administering the placental product of any of claims 1-103 to the subject.
 132. A method of treating a wound comprising providing at least one factor that is a chemotactic factor or a growth factor to the wound by administering the placental product of any of claims 1-103 to the subject.
 133. A method of providing stem cells to tissue of a subject comprising administering the placental product of any of claims 1-103 to the subject. 